Mucin Protein Expression Research Articles

Paeoniflorigenone inhibits ovarian cancer metastasis through targeting the MUC1/Wnt/β‑catenin pathway.

Ovarian cancer (OC) is one of the most common gynecological malignancies. Currently, chemoradiotherapy is the primary clinical treatment approach for OC; however, it has severe side effects and a high rate of recurrence. Thus, there is an urgent need to develop innovative therapeutic options. Paeoniflorigenone (PFG) is a monoterpene compound isolated from the traditional Chinese medicine Paeoniae Radix Rubra. PFG can inhibit the proliferation of tumor cells; however, its anticancer activity against OC has yet to be elucidated. Mucin 1 (MUC1) is highly expressed in various malignant tumors, and is associated with tumor proliferation, metastasis and epithelial‑mesenchymal transition (EMT). In addition, MUC1 affects numerous signaling pathways in tumor cells. In order to develop a possible treatment approach for metastatic OC, the antitumor activity of PFG in OC cells was investigated using Cell Counting Kit‑8 assay, Edu assay, flow cytometry, Transwell assay and western blot analysis. In addition, it was assessed how PFG affects MUC1 expression and function. The experiments revealed that PFG significantly inhibited OC cell proliferation, migration, invasion and EMT. PFG also induced S‑phase cell cycle arrest in OC cells. Furthermore, PFG inhibited MUC1 promoter activity, which led to a decrease in MUC1 protein expression. By contrast, MUC1 promoted OC progression, including cell proliferation, cell cycle progression and cell migration. Stable knockdown of MUC1 in OC cells improved the ability of PFG to block the Wnt/β‑catenin pathway, and to limit tumor cell invasion and migration, whereas MUC1 overexpression partially counteracted the antitumor effects of PFG. In conclusion, the present study demonstrated that PFG may inhibit the MUC1/Wnt/β‑catenin pathway to induce anti‑metastatic, anti‑invasive and anti‑EMT effects on OC. Notably, MUC1 may be a direct target of PFG. Thus, PFG holds promise as a specific antitumor agent for the treatment of OC.

Aryl Hydrocarbon Receptor Regulates Muc2 Production Independently of IL-22 during Colitis.

We previously reported that an aryl hydrocarbon receptor (AhR) ligand, indole-3-carbinol (I3C), was effective at reducing colitis severity through immune cell-mediated interleukin-22 (IL-22) production. Intestinal epithelial cells (IECs) are also involved in regulating colitis, so we investigated their AhR-mediated mechanisms in the current report. A transcriptome analysis of IECs in wildtype (WT) mice revealed that during colitis, I3C regulated select mucin proteins, which could be attributed to goblet cell development. To address this, experiments under in vivo colitis (mice) or in vitro colon organoid conditions were undertaken to determine how select mucin proteins were altered in the absence or presence of AhR in IECs during I3C treatment. Comparing WT to IEC-specific AhR knockout mice (AhRΔIEC), the results showed that AhR expression was essential in IECs for I3C-mediated protection during colitis. AhR-deficiency also impaired mucin protein expression, particularly mucin 2 (Muc2), independently of IL-22. Collectively, this report highlights the important role of AhR in direct regulation of Muc2. These results provide justification for future studies aimed at determining how AhR might regulate select mucins through mechanisms such as direct transcription binding to enhance production.

Krebs von den Lungen 6 (KL-6) is a novel diagnostic and prognostic biomarker in pleural mesothelioma

ObjectivesPleural mesothelioma (PM) is a rare disease with dismal outcome. Systemic treatment options include chemotherapy and immunotherapy, but biomarkers for treatment personalization are missing. The only FDA-approved diagnostic biomarker is the soluble mesothelin-related protein (SMRP). Krebs von den Lungen-6 (KL-6) is a human mucin 1 (MUC1) glycoprotein, which has shown diagnostic and prognostic value as a biomarker in other malignancies. The present study investigated whether KL-6 can serve as a diagnostic and/or prognostic biomarker in PM. Materials and methodsUsing a fully-automated chemiluminescence enzyme immunoassay (CLEIA) for KL-6 and SMRP, pleural effusion samples from 87 consecutive patients with PM and 25 patients with non-malignant pleural disorders were studied. In addition, KL-6 and SMRP levels were determined in corresponding patient sera, and in an independent validation cohort (n = 122). MUC1 mRNA and protein expression, and KL-6 levels in cell line supernatants were investigated in PM primary cell lines in vitro. ResultsPM patients had significantly higher KL-6 levels in pleural effusion than non-malignant controls (AUC 0.78, p < 0.0001). Among PM patients, levels were highest in those with epithelioid or biphasic histologies. There was a strong positive correlation between pleural effusion levels of KL-6 and SMRP (p < 0.0001). KL-6 levels in sera similarly associated with diagnosis of PM, however, to a lesser extent (AUC 0.71, p = 0.008). PM patients with high pleural effusion KL-6 levels (≥303 IU/mL) had significantly better overall survival (OS) compared to those with low KL-6 levels (HR 0.51, p = 0.004). Congruently, high tumor cell MUC1 mRNA expression in primary cell lines associated with prolonged corresponding patient OS (HR 0.35, p = 0.004). These findings were confirmed in an independent validation cohort. ConclusionThis is the first study demonstrating KL-6 as a potential novel liquid-based diagnostic and prognostic biomarker in PM.

Activation of Vitamin D/VDR Signaling Reverses Gemcitabine Resistance of Pancreatic Cancer Cells Through Inhibition of MUC1 Expression.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis due to its therapeutic resistance. Inactivation of vitamin D/vitamin D receptor (VDR) signaling may contribute to the malignant phenotype of PDA and altered expression of oncoprotein mucin 1 (MUC1) may be involved in drug resistance of cancer cells. To determine whether vitamin D/VDR signaling regulates the expression and function of MUC1 and its effect on acquired gemcitabine resistance of pancreatic cancer cells. Molecular analyses and animal models were used to determine the impact of vitamin D/VDR signaling on MUC1 expression and response to gemcitabine treatment. RPPA analysis indicated that MUC1 protein expression was significantly reduced in human PDA cells after treatment with vitamin D3 or its analog calcipotriol. VDR regulated MUC1 expression in both gain- and loss-of-function assays. Vitamin D3 or calcipotriol significantly induced VDR and inhibited MUC1 expression in acquired gemcitabine-resistant PDA cells and sensitized the resistant cells to gemcitabine treatment, while siRNA inhibition of MUC1 was associated with paricalcitol-associated sensitization of PDA cells to gemcitabine treatment in vitro. Administration of paricalcitol significantly enhanced the therapeutic efficacy of gemcitabine in xenograft and orthotopic mouse models and increased the intratumoral concentration of dFdCTP, the active metabolite of gemcitabine. These findings demonstrate a previously unidentified vitamin D/VDR-MUC1 signaling axis involved in the regulation of gemcitabine resistance in PDA and suggests that combinational therapies that include targeted activation of vitamin D/VDR signaling may improve the outcomes of patients with PDA.

배양된 인간 기도 상피세포에서 포볼 에스터로 유발된 뮤신 유전자 발현과 세포 내 NF-κB 신호전달 경로에 대한 하이퍼로사이드의 작용

We investigated whether hyperoside, an anti-inflammatory natural product, significantly affects phorbol 12- myristate 13-acetate (PMA)-induced airway expression of MUC5AC encoded mucin. Confluent NCI-H292 cells were pretreated with hyperoside for 30 min, then stimulated with PMA for 24 h or the indicated periods. MUC5AC mucin mRNA expression was measured by RT-PCR. MUC5AC mucin protein expression was quantitated by ELISA. To elucidate the mechanism of hyperoside action, we investigated the effect of hyperoside on PMA-induced NF-κB signaling by western blot analysis. The results were as follows: (1) Hyperoside significantly inhibited MUC5AC mucin protein production and down-regulated MUC5AC mucin mRNA expression; (2) Hyperoside inhibited PMA-induced activation (phosphorylation) of inhibitory kappa B kinase (IKK), thereby inhibiting phosphorylation and degradation of inhibitory kappa Bα (IκBα); (3) Hyperoside inhibited PMA-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65, thereby down-regulating MUC5AC mucin gene expression in NCI-H292 cells. These results suggest that hyperoside can regulate mucin gene expression by airway epithelial cells through regulation of the NF-κB signaling pathway.

Novel insights into the roles and therapeutic implications of MUC1 oncoprotein via regulating proteins and non-coding RNAs in cancer.

Mucin 1 (MUC1) is a heterodimeric transmembrane glycoprotein that protects epithelial cells in mammals. The transmembrane C-terminal subunit (MUC1-C) plays a crucial role in oncogenesis. As an oncoprotein, MUC1-C regulates a number of proteins that are associated with tumorigenesis by interacting with oncoproteins, transcription factors, coactivators, etc., inducing proliferation, epithelial-mesenchymal transition (EMT), invasion, stemness, immune evasion, and drug resistance. Moreover, MUC1-C modulates the expression of non-coding RNAs (ncRNAs), which further regulate carcinogenesis by directly binding to specific proteins. ncRNAs can also affect MUC1 protein expression by targeting the MUC1 mRNA 3′ untranslated region (UTR). A series of ncRNAs can modulate cancer development by regulating MUC1-C. This review focuses on the interaction of MUC1-C with proteins and ncRNAs in cancer progression. We also summarize the recent advances in immunotherapy with a focus on therapeutic approaches based on MUC1-C and nanocarrier complexes for cancer treatment.

The mucin protein MUCL1 regulates melanogenesis and melanoma genes in a manner dependent on threonine content.

SummaryBackgroundThe regulation of melanogenesis has been investigated as a long‐held aim for pharmaceutical manipulations with denotations for malignancy of melanoma. Mucins have a protective function in epithelial organs; however, in the most outer organ, the skin, the role of mucins has not been studied enough.ObjectivesOur initial hypothesis developed from the identification of correlations between pigmentation and expressions of skin mucins, particularly those existing in skin tissue. We aimed to investigate the action of mucins in human melanocytic cells.Materials and methodsThe expression of mucin proteins in human skin was investigated using microarray data from the Human Protein Atlas consortium (HPA) and the Genotype‐Tissue Expression consortium (GTEx) database. Mucin expression was measured at RNA and protein levels in melanoma cells. The findings were further validated and confirmed by analysis of independent experiments.ResultsWe found that the several mucin proteins showed expression in human skin cells and among these, mucin‐like protein 1 (MUCL1) showed the highest expression and also clear negative correlation with melanogenesis in epidermal melanocytes. We confirmed the correlations between melanogenesis and MUCL1 by revealing negative correlations in melanocytes with different melanin production, resulting from increased composition of threonine, mucin‐conforming amino acid, and increased autophagy‐related forkhead‐box O signalling. Furthermore, threonine itself affects melanogenesis and metastatic activity in melanoma cells.ConclusionsWe identified a significant association between MUCL1 and threonine with melanogenesis and metastasis‐related genes in melanoma cells. Our results define a novel mechanism of mucin regulation, suggesting diagnostic and preventive roles of MUCL1 in cutaneous melanoma.

Steroidal but not embryonic regulation of mucin 1 expression in bovine endometrium.

In cow herd management, inadequate embryo implantation leads to pregnancy loss and causes severe economic losses. Thus, it is crucial to understand the molecular mechanisms underlying endometrial receptivity and subsequent embryo implantation. Transmembrane glycocalyx mucin 1 (MUC1) has a large and highly glycosylated extracellular domain known to inhibit embryo implantation via steric hindrance. The role of MUC1 in the bovine endometrium remains to be explored. Herein, we used simple but reliable in vivo and in vitro experiments to investigate the expression and regulation of MUC1 in the bovine endometrium. MUC1 gene expression was analyzed in endometrial epithelial cells collected by the cytobrush technique using reverse transcription-quantitative polymerase chain reaction. MUC1 protein expression was evaluated by immunohistochemical analysis of endometrial samples collected from slaughtered cows. We used an in vitro cell culture model to study the regulation of MUC1 expression by treating cells with sex steroidal hormones or co-culturing cells with a blastocyst. The results revealed that MUC1 was expressed and localized to the apical surface of luminal epithelial cells in the bovine endometrium. MUC1 expression disappeared during the luteal phase of the estrous cycle and during pregnancy. 17β-estradiol induced MUC1 expression, whereas progesterone inhibited its increase and co-culturing with blastocysts did not affect the expression. A long postpartum interval is a known risk factor for reduced fertility, and MUC1 expression was higher in this compromised condition. Our results demonstrated the MUC1 regulation by steroid hormones in bovine endometrium for embryo implantation, and we observed a negative correlation between MUC1 expression and fertility.

GUAIFENESIN MODIFIES AIRWAY MUCUS, REDUCES MUCUS-CELL SURFACE INTERACTIONS, AND INCREASES MUCOCILIARY TRANSPORT IN AN ORGANOTYPIC IN VITRO MODEL OF MUCUS HYPERSECRETION

SESSION TITLE: Phenotyping and Management of COPD SESSION TYPE: Original Investigations PRESENTED ON: October 18-21, 2020 PURPOSE: Airway mucus hypersecretion is a hallmark of chronic bronchitis (CB) and is strongly associated with cough discomfort, reduced pulmonary function, recurring respiratory infections, and the development of obstructive lung disease. Guaifenesin (GGE) is currently the only FDA-approved oral expectorant and the most common active ingredient in prescription and over-the-counter medicines for treatment of acute respiratory congestion and stable CB. Studies in patients with CB demonstrate that GGE reduces airway secretion viscoelasticity and mucus adhesion, inhibits cough reflex sensitivity, and increases mucociliary clearance (MCC). However, despite its broad use, the mechanism of action for GGE remains unclear. Here, we investigate whether GGE acts directly on the respiratory epithelium to modify epithelial cell secretions, reduce mucus/mucosa interactions, and enhance mucociliary transport (MCT) using an organotypic in vitro model of mucus hypersecretion. METHODS: Primary human bronchial epithelial cells (hBECs) were collected from healthy (HC), heavy smoker (HS; 48.3±18.9 pack year), and COPD (51.3±10.3 pack year) donor lungs (N=3 per group), and differentiated at an air-liquid interface to a mucociliary phenotype. At culture day 35, 20µg/ml GGE was supplemented to the basolateral medium and cultures were maintained in the presence of drug for an additional 15 days. Apical secretions were allowed to accumulate for the duration of the 15-day treatment. Following treatment, mucus concentration, mucus/mucosa interaction strength, in vitro MCT, ciliary beat frequency (CBF), and mucin protein expression were compared. RESULTS: Apical secretions collected from HS and COPD cultures had a higher mucus concentration (12.3±3.0 and 11.1±2.2 % solids, respectively) than HC cultures (7.6±1.1). Moreover, increased mucus concentration was associated with greater mucus/mucosa interaction strength and lower baseline MCT as compared to HC. In all donor groups, 15-day GGE treatment reduced mucus concentration (? % solids: 2.2±1.2 HC, 1.9±0.6 HS, and 2.4±1.8 COPD) and mucus/mucosa interaction strength (? mN/cm: 1.8±0.4 HC, 3.6±1.9 HS, and 1.6±0.4 COPD). GGE-mediated reductions in mucus concentration and mucus/mucosa interaction strength translated to increased in vitro MCT (? µm/sec: 35.1±21.9 HC, 19.6±30.1 HS, and 4.7±0.8 COPD). GGE treatment did not significantly alter MUC5AC or MUC5B secreted protein levels, or CBF. CONCLUSIONS: Our findings indicate that GGE significantly reduces airway mucus concentration, reduces mucus-cell surface interaction strength, and increases MCT in vitro through direct interactions with airways epithelial cells in all donor groups (HC, HS, and COPD). CLINICAL IMPLICATIONS: These data suggest that GGE may act directly at the level of the respiratory epithelium to modify airway mucus and improve MCC in conditions characterized by mucus hypersecretions. DISCLOSURES: Global Medical Lead Respiratory relationship with Reckitt Benckiser Please note: $1001 - $5000 Added 04/10/2020 by Graça Maria Alves da Cunha Coutinho, source=Web Response, value=Salary No relevant relationships by Brian Button, source=Web Response Receive research support relationship with Reckitt Benckiser Please note: >$100000 Added 04/09/2020 by Phillip Clapp, source=Web Response, value=Grant/Research Support No relevant relationships by Miriam Figueira, source=Web Response no disclosure on file for Ly Nguyen; Employee relationship with Rekitt Benckiser Please note: >$100000 Added 06/11/2020 by Karin Nicholson, source=Web Response, value=Salary No relevant relationships by Timother Shea, source=Web Response No relevant relationships by Holly Thacker, source=Web Response

422 Positive Association Between CDX2 and MUC2 Intestinal Factors and Dysplasia in Barrett’s Esophagus Utilizing WATS3D Tissue Sampling and Analysis

INTRODUCTION: During the pathogenesis of Barrett’s esophagus (BE), the background columnar epithelium often shows evidence of intestinal differentiation, such as early expression of intestinal specific transcription factors (CDX2) and late expression of mucin proteins (MUC2), prior to the development of goblet cells. The clinical significance of these markers is unknown. The aim of this study was to evaluate the association between the expression of CDX2 and MUC2, and the presence and grade of dysplasia in a large cohort of patients with endoscopic evidence of esophageal columnar mucosa using wide area transepithelial samples (WATS3D, CDx Diagnostics, Suffern, NY). METHODS: Over a 2-year period, we collected data from 129,158 consecutive patients with endoscopic evidence of esophageal salmon colored mucosa who were evaluated with WATS: 125,749 no dysplasia (ND), 2,012 crypt dysplasia (CD), 745 low grade dysplasia (LGD), and 652 high grade dysplasia/adenocarcinoma (HGD/EAC). CDX2 and MUC2 immunostaining was performed, and then their extent was graded semi-quantitatively (on a 4-point scale) in the background non-dysplastic, non-goblet epithelium, from all patients. The CDX2 and MUC2 results were then correlated statistically with the presence and grade of dysplasia. RESULTS: Table 1 summarizes the data according to dysplasia grade. Overall, 33.3% of cases were CDx2- MUC2-, 30.6% were CDx2+ MUC2-, and 36.1% were CDX2+ MUC2+. A significant (P &lt; 0.0001) increase in CDX2+ MUC2+ staining occurred in dysplasia cases (97.0%) compared to non- dysplasia cases (34.5%). Upon regression analysis, there was a highly significant (P &lt; 0.0001) association between both the presence and grade of CDX2 and MUC2 staining and dysplasia, that increased progressively from CD to LGD to HGD/EAC. CDX2+ MUC2+ was present, uniformly, in more than 94% of all dysplasia categories. In a sub-analysis of either CDX2 or MUC2 staining in ND vs. HGD/EAC cases only, there was a significant increase (P &lt; 0.0001) in the grade of CDX2, and in the grade of MUC2, staining, such that 3+ or 4+ CDX2 or MUC2 staining was noted in 8.8% and 9.1% of ND cases, respectively, compared to 54% and 41%, respectively, in HGD/EAC. CONCLUSION: CDX2 and/or MUC2 positivity is a highly significant risk factor for dysplasia/EAC in patients with endoscopic evidence of esophageal columnar mucosa. CDX2/MUC2 immunostaining may represent a more effective method of confirming Barrett’s esophagus, and the risk for dysplasia/cancer, than the identification of goblet cells.

Endometrial Alterations After the Transfer of Embryonic Remains

In this study, we investigated the expression of mucin 1 (MUC1) mRNA and protein in sheep endometrium at different time points during follicular and luteal phases of estrous cycle, and also determined the effect of steroid hormone treatments and interferon tau (IFNτ) on MUC1 mRNA expression in endometrial cell culture in vitro. In experiment one, 15 Welsh mountain ewes were synchronized to a common estrus and killed at precise stages of estrous cycle corresponding to (1) pre-LH peak, (2) LH peak, (3) post-LH peak, (4) early luteal, and (5) mid-luteal. Reproductive tracts were harvested and mRNA was extracted from the endometrial tissues. Parts of the uterine horns were fixed for immunohistochemistry. In experiment two, mixed populations of ovine endometrial cells (from slaughterhouse material collected at the postovulatory stage of the estrous cycle) were cultured to 70% confluence before treatment with (1) progesterone (P4, 10 ng/mL, for 48 hours), (2) estradiol (E2, 100 pg/mL, for 48 hours), or with (3) E2 priming for 12 hours (100 pg/mL) followed by P4 (10 ng/mL) for 36 hours. These were compared with: (4) IFNτ (10 ng/mL, for 48 hours), and (5) basic medium (Dulbecco Modified Eagle Medium /F12) as control. The results showed that MUC1 mRNA and protein expression in sheep endometrium were highest during the midluteal stage and very low during the post-LH period compared with the other stages (P < 0.05). MUC1 immunostaining in the luminal epithelium was apically restricted and was not significantly different across all stages of estrous cycle except at the post-LH peak where it was significantly low. In cell culture, MUC1 mRNA expression was significantly upregulated by both steroids either singly or in combination (P < 0.05), and downregulated in the presence of IFNτ. In conclusion, endometrial MUC1 expression is cyclically regulated by both E2 and P4 in vivo and in vitro, and directly downregulated by IFNτ treatment in vitro.

Effects of Topical Mucolytic Agents on the Tears and Ocular Surface: A Plausible Animal Model of Mucin-Deficient Dry Eye.

A topical mucolytic agent, N-acetylcysteine (NAC), has been used to create an animal model without the intestinal mucus layer. In this study, we investigated the effects of topical NAC on the tears and ocular surface. NAC-treated models were established by topically administering 10% NAC four times daily for 5 days in male Sprague-Dawley rats. Clinical parameters and the expression of mucin proteins and genes were evaluated. Alterations in the conjunctival epithelium and goblet cells were observed. The NAC group showed significant decreases in tear secretion, corneal wetting ability, tear MUC5AC concentration, and conjunctival goblet cell numbers as compared with the control group (all P < 0.01). In addition, significant increases in corneal fluorescein score and rose bengal scores were observed in the NAC group versus in the control group (P < 0.05 and P < 0.01, respectively). Hematoxylin and eosin (H&E) staining and scanning electron microscopy clearly showed damage in the epithelial cell layer and microvilli of the NAC group. Although there was no significant difference in MUC16 gene expression, the MUC16 concentration of the tear film and ocular surface tissue was significantly increased in the NAC group versus in the control group (P < 0.01 and P < 0.05, respectively). Five-day treatment with 3% diquafosol had minimal therapeutic effect in NAC-treated rat eyes. Topical administration of 10% NAC induced ocular surface damage and tear film instability by prompting MUC16 disruption and release from the ocular surface. This animal model could be used to study dry eye disease, especially the mucin-deficiency subtype.

Expression and function of MUC1 in uterine tissues during early pregnancy in sheep after natural oestrous or artificially-induced oestrous

Mucin 1 (MUC1), a cell surface glycoprotein, is expressed mainly in the endometrial luminal epithelium (LE) and glandular epithelium (GE) of the endometrium in many mammalian species including mice, rats, pigs, sheep, horses and humans during various stages of a menstrual or oestrous cycle, where it plays an important role in embryo implantation and placentation. However, the expression and function of MUC1 in uterine tissues during early pregnancy in sheep after artificially-induced oestrous is not known. Therefore, we investigated the expression and function of MUC1 in the early pregnant and non-pregnant uterine tissues of sheep with natural oestrous or artificially-induced oestrous on days 10, 12, 14, 16 and 18 of the cycle by in situ hybridization, quantitative real-time polymerase chain reaction, immunohistochemical staining and western blotting methods. According to our results, MUC1 mRNA and protein expression increased initially but then decreased from days 10–18, peaking on day 14 in the uterine tissues of non-pregnant ewes after both natural and artificially-induced oestrous. MUC1 protein localisation was observed in the LE on days 10, 12 and 14 and in the GE on days 16 and 18. In contrast, MUC1 mRNA and protein expression increased on days 10 and 12, decreased on day 14, but increased again on days 16 and 18 in the uterine tissues of pregnant ewes both in natural oestrous and in artificially-induced oestrous. Additionally, the MUC1 mRNA and protein expression levels in the uterine tissues of the early pregnant sheep were significantly lower than those in the non-pregnant sheep on days 10, 14, and 18, except on day 16(P < 0.01). Enhancing MUC1 protein expression with oestrogen or/and progesterone decreased the blastocyst adhesion rate when blastocysts were co-cultured with endometrial epithelial cells (EECs), while inhibiting MUC1 protein expression with IFN-τ increased the blastocyst adhesion rate when the blastocysts were co-cultured with EECs. Compared with the ewes undergoing natural oestrus, the expression trend and regulation of MUC1 did not change, and the MUC1 expression levels only increased under artificial oestrus conditions. Our data provide important information for improving the conception rate in sheep undergoing artificially-induced oestrus and offer some reference points relating to embryo transfer, oestrus synchronisation and superovulation.

Clinicopathological implications to micropapillary bladder urothelial carcinoma of the presence of sialyl Lewis X-decorated mucin 1 in stroma-facing membranes

ObjectivesBladder urothelial carcinoma (UC) comprises more than 90% of all bladder cancers. Among several UC variants, micropapillary UC (MPUC) is a rare one with high potential for lymphovascular invasion and subsequent lymph node metastasis. Histologically, MPUC is characterized by the presence of small papillary carcinoma cell clusters surrounded by lacunar spaces. Immunohistochemically, the outer circumference of these clusters, that is, the stroma-facing membrane of carcinoma cells, is reportedly almost invariably positive for mucin 1 (MUC1) protein and to a lesser extent for sialyl Lewis X (sLeX) carbohydrates; however, the clinicopathological implications of these expression patterns have not been fully investigated. Materials and methodsWe performed immunohistochemical analysis of MPUC (n = 11) and conventional UC (n = 57) for MUC1 and sLeX to determine whether these factors immunolocalized. Dual immunofluorescence staining was also carried out to assess MUC1 and sLeX colocalization. We also performed Western blot analysis of Chinese hamster ovary cells misexpressing both recombinant epitope-tagged MUC1 and glycosyltransferases enabling sLeX biosynthesis. ResultsMPUC samples preferentially exhibited both MUC1 protein and sLeX carbohydrate expression on the stroma-facing membrane of carcinoma cells. Based on univariate analysis, MUC1 expression in that pattern was positively correlated with tumor extension, lymphovascular invasion, lymph node metastasis, disease stage, and relatively poor patient prognosis. A comparable sLeX expression pattern also correlated positively with tumor extension and nodal metastasis. Based on multivariate analysis, localization of MUC1 and sLeX on the stroma-facing side of the membrane was positively correlated with lymph node metastasis. ConclusionsOverall, our immunofluorescence findings as well as immunoprecipitation analyses of Chinese hamster ovary cell transfectants strongly suggest that MUC1 is a potential scaffold protein for sLeX carbohydrates in MPUC. Both MUC1 and sLeX may cooperatively contribute to MPUC histogenesis and clinicopathological characteristics.

Mucin-1 correlates with survival, smoking status, and growth patterns in lung adenocarcinoma.

Mucin-1 (MUC1) affects cancer progression in lung adenocarcinoma, and its aberrant expression pattern has been correlated with poor tumor differentiation and impaired prognosis. In this study, the immunohistochemical expression of MUC1 and Mucin-4 (MUC4) was analyzed in a series of 106 surgically operated stage I-IV pulmonary adenocarcinomas. MUC1 immunohistochemistry was evaluated according to the Nagai classification, and the immunohistochemical profile of the tumors was correlated with detailed clinical and histological data. The effect of cigarette smoke on MUC1 expression in lung cancer cell lines was examined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoelectron microscopy (IEM). In contrast to the normal apical localization of MUC1, a basolateral and cytoplasmic (depolarized) MUC1 expression pattern was frequently encountered in the high-grade subtypes, i.e., solid predominant adenocarcinoma and the cribriform variant of acinar predominant adenocarcinoma (p<0.001), and was rarely observed in tumors containing a non-predominant lepidic component (p<0.001). Furthermore, the altered staining pattern of MUC1 correlated with stage (p=0.002), reduced overall survival (p=0.031), and was associated with smoking (p<0.001). When H1650 adenocarcinoma cells were exposed to cigarette smoke and analyzed by RT-qPCR and IEM, the levels of the MUC1 transcript and protein were elevated (p=0.042). In conclusion, MUC1 participates in the pathogenesis of lung adenocarcinoma and associates with smoking both in vitro and in vivo. In lung adenocarcinoma, depolarized MUC1 protein expression correlated with histological growth patterns, stage, and patient outcome.

In vivo and in vitro studies of MUC1 regulation in sheep endometrium

In this study, we investigated the expression of mucin 1 (MUC1) mRNA and protein in sheep endometrium at different time points during follicular and luteal phases of estrous cycle, and also determined the effect of steroid hormone treatments and interferon tau (IFNτ) on MUC1 mRNA expression in endometrial cell culture in vitro. In experiment one, 15 Welsh mountain ewes were synchronized to a common estrus and killed at precise stages of estrous cycle corresponding to (1) pre-LH peak, (2) LH peak, (3) post-LH peak, (4) early luteal, and (5) mid-luteal. Reproductive tracts were harvested and mRNA was extracted from the endometrial tissues. Parts of the uterine horns were fixed for immunohistochemistry. In experiment two, mixed populations of ovine endometrial cells (from slaughterhouse material collected at the postovulatory stage of the estrous cycle) were cultured to 70% confluence before treatment with (1) progesterone (P4, 10 ng/mL, for 48 hours), (2) estradiol (E2, 100 pg/mL, for 48 hours), or with (3) E2 priming for 12 hours (100 pg/mL) followed by P4 (10 ng/mL) for 36 hours. These were compared with: (4) IFNτ (10 ng/mL, for 48 hours), and (5) basic medium (Dulbecco Modified Eagle Medium /F12) as control. The results showed that MUC1 mRNA and protein expression in sheep endometrium were highest during the midluteal stage and very low during the post-LH period compared with the other stages (P < 0.05). MUC1 immunostaining in the luminal epithelium was apically restricted and was not significantly different across all stages of estrous cycle except at the post-LH peak where it was significantly low. In cell culture, MUC1 mRNA expression was significantly upregulated by both steroids either singly or in combination (P < 0.05), and downregulated in the presence of IFNτ. In conclusion, endometrial MUC1 expression is cyclically regulated by both E2 and P4in vivo and in vitro, and directly downregulated by IFNτ treatment in vitro.

Lynch syndrome-associated breast cancers do not overexpress chromosome 11-encoded mucins

Mismatch repair-deficient breast cancers may be identified in Lynch syndrome mutation carriers, and have clinicopathological features in common with mismatch repair-deficient colorectal and endometrial cancers such as tumour-infiltrating lymphocytes and poor differentiation. Mismatch repair-deficient colorectal cancers frequently show mucinous differentiation associated with upregulation of chromosome 11 mucins. The aim of this study was to compare the protein expression of these mucins in mismatch repair-deficient and -proficient breast cancers. Cases of breast cancer (n=100) were identified from families where (1) both breast and colon cancer co-occurred and (2) families met either modified Amsterdam criteria or had at least one early-onset (<50 years) colorectal cancer. Tumour sections were stained for the epithelial mucins, MUC2, MUC5AC, MUC5B and MUC6, and the homeobox protein CDX2, a regulator of MUC2 expression. In all, 16 mismatch repair-deficient Lynch syndrome breast cancers and 84 non-Lynch breast cancers were assessed for altered mucin expression. No significant difference in the expression of MUC2, MUC5AC or MUC6 was observed between the mismatch repair-deficient and mismatch repair-proficient breast cancers; however, there was a trend for mismatch repair-deficient tumours to express high levels of MUC5B less frequently (P=0.07, OR=0.2 (0.0–1.0)). Co-expression of two or more gel-forming mucins was common. Ectopic expression of CDX2 was associated with expression of MUC2 (P=0.035, OR=8.7 (1.3–58.4)). Mismatch repair-deficient breast cancers do not show differential expression of the mucins genes on chromosome 11 when compared with mismatch repair-proficient breast cancers, in contrast with mismatch repair-deficient colorectal and endometrial cancers, which frequently have increased mucin protein expression when compared with their mismatch repair-proficient counterparts. In addition, ectopic CDX2 expression is positively associated with de novo MUC2 expression.

Aberrant Expression of Mucin Core Proteins and O-Linked Glycans Associated with Progression of Pancreatic Cancer

Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed in pancreatic adenocarcinoma-sialyl Tn (STn), Tn, T antigen, sialyl Lewis A (CA19-9), sialyl Lewis C (SLeC), Lewis X (LeX), and sialyl LeX (SLeX)-during the progression of pancreatic cancer from early stages to metastatic disease. Immunohistochemical analyses of mucin and associated glycan expression on primary tumor and liver metastatic tumor samples were conducted with matched sets of tissues from 40 autopsy patients diagnosed with pancreatic adenocarcinoma, 14 surgically resected tissue samples, and 8 normal pancreata. There were significant changes in mucin expression patterns throughout disease progression. MUC1 and MUC4 were differentially glycosylated as the disease progressed from early pancreatic intraepithelial neoplasias to metastatic disease. De novo expression of several mucins correlated with increased metastasis indicating a potentially more invasive phenotype, and we show the expression of MUC6 in acinar cells undergoing acinar to ductal metaplasia. A "cancer field-effect" that included changes in mucin protein expression and glycosylation in the adjacent normal pancreas was also seen. There are significant alterations in mucin expression and posttranslational processing during progression of pancreatic cancer from early lesions to metastasis. The results are presented in the context of how mucins influence the biology of tumor cells and their microenvironment during progression of pancreatic cancer.

Mutation spectrum in human colorectal cancers and potential functional relevance

BackgroundSomatic variants, which occur in the genome of all cells, are well accepted to play a critical role in cancer development, as their accumulation in genes could affect cell proliferations and cell cycle.MethodsIn order to understand the role of somatic mutations in human colorectal cancers, we characterized the mutation spectrum in two colorectal tumor tissues and their matched normal tissues, by analyzing deep-sequenced transcriptome data.ResultsWe found a higher mutation rate of somatic variants in tumor tissues in comparison with normal tissues, but no trend was observed for mutation properties. By applying a series of stringent filters, we identified 418 genes with tumor specific disruptive somatic variants. Of these genes, three genes in mucin protein family (MUC2, MUC4, and MU12) are of particular interests. It has been reported that the expression of mucin proteins was correlated with the progression of colorectal cancer therefore somatic variants within those genes can interrupt their normal expression and thus contribute to the tumorigenesis.ConclusionsOur findings provide evidence of the utility of RNA-Seq in mutation screening in cancer studies, and suggest a list of candidate genes for future colorectal cancer diagnosis and treatment.

Improving risk stratification in superficial (T1) gastroesophageal adenocarcinoma: Association of the intestinal phenotype (MUC2 positive) with prognosis.

33 Background: An increasing proportion of esophageal and gastroesophageal junction adenocarcinomas (EAC) are limited to the superficial mucosal layer (stage classification T1) at the time of diagnosis. Intramucosal lesions (stage classification T1a) are increasingly managed with endoscopic resection due to their relatively low risk of nodal metastasis and favorable prognosis compared to those with submucosal invasion (stage classification T1b). At esophagectomy, however, the majority of T1b EAC are node-negative; conversely, a small proportion of T1a EAC will have nodal metastasis at esophagectomy and/or develop recurrent disease. Additional prognostication tools, such as tumor biomarker panels, are needed to optimize treatment algorithms. Known to carry prognostic significance in other tumors, we evaluated mucin protein expression to determine whether the mucin phenotype in early EAC was associated with survival outcomes. Methods: We classified 141 induction naive, resected, T1 EAC as intestinal-, gastric- or ductal-type depending on the expression pattern of gastrointestinal mucins (MUC2, MUC5AC, MUC6 and MUC1) determined by immunohistochemistry (IHC) on tissue microarrays. CDX2 expression was assessed by IHC and EGFR amplification was assessed by in situ hybridization. The association between survival and mucin subtypes was determined in univariate and multivariate analyses. Results: The intestinal phenotype (MUC2+) is associated with poor prognosis in T1b (HR 2.1, 95%CI 1.1-4.0) and node negative T1 EAC (HR 2.5, 95%CI 1.1-5.7) after adjusting for other significant prognostic factors. Intestinal type EAC also showed accelerated time to first recurrence in these subgroups. The colloid histologic subtype and CDX2 expression were positively associated with MUC2 expression, but EGFR amplification was not. Conclusions: The mucin phenotype may serve as a useful adjunct to conventional risk assessment for optimizing therapy for potentially resectable, superficial EAC.