Mucin Research Articles

Chemoenzymatic Synthesis of Glycopeptides to Explore the Role of Mucin 1 Glycosylation in Cell Adhesion.

Post-translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O-glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell-adhesion experiments with MCF-7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.

Superparamagnetic iron oxide nanoparticles target BxPC-3 cells and silence MUC4 for the treatment of pancreatic cancer

PurposeSuperparamagnetic iron oxide nanoparticles (SPION) are excellent magnetic resonance imaging (MRI) contrast agents. Mucin 4 (MUC4) acts as pancreatic cancer (PC) tumor antigen and influences PC progression. Small interfering RNAs (siRNAs) are used as a gene-silencing tool to treat a variety of diseases. MethodsWe designed a therapeutic probe based on polyetherimide-superparamagnetic iron oxide nanoparticles (PEI-SPION) combined with siRNA nanoprobes (PEI-SPION-siRNA) to assess the contrast in MRI. The biocompatibility of the nanocomposite, and silencing of MUC4 were characterized and evaluated. ResultsThe prepared molecular probe had a particle size of 61.7 ± 18.5 nm and a surface of 46.7 ± 0.8mV and showed good biocompatibility in vitro and T2 relaxation efficiency. It can also load and protect siRNA. PEI-SPION-siRNA showed a good silencing effect on MUC4. ConclusionPEI-SPION-siRNA may be beneficial as a novel theranostic tool for PC.

Canonical and truncated transglutaminase-2 regulate mucin-1 expression and androgen independency in prostate cancer cell lines

Androgen independency is associated with poor prostate cancer (PCa) survival. Here we report that silencing of transglutaminase-2 (TG2) expression by CRISPR-Cas9 is associated with upregulation of androgen receptor (AR) transcription in PCa cell lines. Knockout of TG2 reversed the migratory potential and anchorage independency of PC3 and DU145 cells and revealed a reduced level of mucin-1 (MUC1) RNA transcript through unbiased multi-omics profiling, which was restored by selective add-back of the truncated TG2 isoform (TGM2_v2). Silencing of AR resulted into increased MUC1 in TG2KO PC3 cells showing that TG2 affects transcriptional regulation of MUC1 via repressing AR expression. Treatment of PC3 WT cell line with TG2 inhibitor ZDON led to a significant increase in AR expression and decrease in MUC1. ZDON also blocked the formation of MUC1-multimers labelled with TG amine-donor substrates in reducing conditions, revealing for the first time a role for TG2, which we show to be externalised via extracellular vesicles, in MUC1 stabilisation via calcium-dependent transamidation. A specific antibody towards TGM2_v2 revealed its restricted nuclear location compared to the canonical long form of TG2 (TGM2_v1), which is predominantly cytosolic, suggesting that this form contributes to the previously suggested TG2-mediated NF-κB activation and AR transcriptional repression. As TGM2_v2 transcription was increased in biopsies of early-stage prostate adenocarcinoma (PRAD) patients compared to subjects presenting inflammatory prostatitis, and total TG2 protein expression significantly increased in PRAD versus normal tissue, the role of TG2 and its truncated form as a prostate malignancy marker is suggested. In conclusion, this investigation has provided the first unbiased discovery of a novel pathway mediated by TG2 via MUC1, which is shown to contribute to androgen insensitivity and malignancy of PCa cells and be upregulated in PCa biopsies, with potential relevance to cancer immune evasion.

Impact of feed additives and host-related factors on bacterial metabolites, mucosal integrity and immune response in the ileum of broilers

The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 one-day-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 × 109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p > 0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p < 0.05). mRNA expression of cytokines, mucin 2 (MUC2) and claudin 5 (CLDN5) was similar; increased from day 7 to 21 and decreased afterward (p < 0.05). Villus height, crypt depth and villus surface area increased with age (p < 0.05). Acidic goblet cells (GC) number and density increased after day 21 (p < 0.05). Ross broilers showed higher D-lactate concentration and IFN-γ expression, while Cobb broilers had greater IL-4, IL-6 and TNF-α expression and higher total GC number (p < 0.05). Female displayed higher villus height and GC number and density (mixed and total GC) than male (p < 0.05). The effect of dietary treatment was not found on any investigated variables (p > 0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system.

MUC1 induces the accumulation of Foxp3+ Treg cells in the tumor microenvironment to promote the growth and metastasis of cholangiocarcinoma through the EGFR/PI3K/Akt signaling pathway.

Tumor microenvironment (TME) plays an important role in the progression of cholangiocarcinoma. This study aims to explore whether Mucin 1 (MUC1) regulates Foxp3+ Treg cells in the TME of cholangiocarcinoma through the epidermal growth factor receptor (EGFR)/phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. High-throughput sequencing dataset in the GEO database combined with GeneCards and Phenolyzer databases was used to obtain key genes in cholangiocarcinoma, followed by downstream pathway prediction. The relationship among MUC1, EGFR, and PI3K/Akt signaling pathway was explored. CD4+ T cells extracted from peripheral blood were induced to differentiate into Treg cells, followed by co-culture with cholangiocarcinoma cells. A mouse model was constructed to detect the role of MUC1 in the accumulation of Foxp3+ Treg cells, malignant phenotypes of cholangiocarcinoma, and tumorigenesis in vivo. MUC1, highly expressed in cholangiocarcinoma, might be involved in cholangiocarcinoma development. MUC1 interacted with the EGFR to activate the EGFR/PI3K/Akt signaling pathway. MUC1 overexpression could activate the EGFR/PI3K/Akt signaling pathway, which promoted the accumulation of Foxp3+ Treg cells in the TME and the malignant phenotypes of cholangiocarcinoma cells both in vitro and in vivo and enhanced tumorigenesis in vivo. MUC1 may interact with EGFR to activate the EGFR/PI3K/Akt signaling pathway, which induces the accumulation of Foxp3+ Treg cells, enhancing the malignant phenotypes of cholangiocarcinoma cells and tumorigenesis in vivo and ultimately augmenting cholangiocarcinoma growth and metastasis.

Serum KL-6 levels predict clinical outcomes and are associated with MUC1 polymorphism in Japanese patients with COVID-19

BackgroundKrebs von den Lungen-6 (KL-6) is a known biomarker for diagnosis and monitoring of interstitial lung diseases. However, the role of serum KL-6 and the mucin 1 (MUC1) variant (rs4072037)...

High-efficiency interparticle DNA walker amplifier combined with N-Ti3C2 quantum dots as electrochemiluminescence emitters for sensitive protein assay

In this work, an electrochemiluminescence (ECL) biosensor based on interparticle DNA walker as effective signal amplification strategy and N-Ti3C2 QDs as high-efficiency emitters was designed for sensitive assay of mucin 1 (MUC1), which is closely related to the occurrence and development of tumors. First, an interparticle DNA walker which contains walking particles (WPs) and orbit particles (OPs) was fabricated. In the presence of target MUC1 and DNAzyme, the WPs can walk along the surface of OPs, and they can continue to walk along next OPs after completing previous walk. As the moving freedom and area improved, the interparticle DNA walker could effectively improve the conversion efficiency of target. Furthermore, N-Ti3C2 QDs with high ECL luminous efficiency were synthesized by using polyethyleneimine (PEI) as nitrogen source. Due to the nitrogen-rich of PEI, the relative ECL quantum efficiency of the proposed N-Ti3C2 QDs was calculated to be 1.5 times higher than our previous reported N-Ti3C2 QDs that utilizing ethylenediamine as nitrogen source. Therefore, taking the advantage of the improved moving freedom and area of the interparticle DNA walker and high ECL efficiency of the proposed N-Ti3C2 QDs, the fabricated ECL biosensor for MUC1 determination displayed a low detection limit with a wide linear range, which provides a potential feasible method for other protein sensitive assay.

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.

Aptamer modified Ti3C2 nanosheets application in smart targeted photothermal therapy for cancer

BackgroundTi3C2 is a type of transition metal carbides and nitrides (MXenes) with high light-to-heat conversion efficiency property, which has been widely used in cancer treatment recently. In fact, active targeting delivery of MXenes nanomaterials with targeting molecule could enhance the therapeutic efficacy. However, targeted therapy of MXenes has not been further studied in the past. Aptamers (Apt) with excellent affinity and high specificity properties have been widely used as targeting tools. Predictably, the incorporation of Apt into Ti3C2 nanomaterials will offer an unprecedented opportunity in the research fields of cancer targeted therapy.ResultsTransmembrane glycoprotein mucin 1 (MUC1) is overexpressed on the surface of MCF-7 cells, and MUC1 Apt (Apt-M) could target MCF-7 cells with high affinity and specificity. Here, a smart targeting nanotherapeutic system Ti3C2/Apt-M was fabricated, which could specifically recognize and enter in MCF-7 cells. Benefitting from the desirable targeted performance of Apt-M, MCF-7 cells completed the ingestion process of Ti3C2/Apt-Mf nanosheets within 4 h, and Apt-M facilitated the entry of the Ti3C2/Apt-Mf nanosheets into MCF-7 cells. Besides, Ti3C2/Apt-M nanosheets exhibited the potential as an outstanding photothermal agent (PTA) because of the photothermal performance inherited from wrapped Ti3C2 nanosheets. As demonstrated, upon 808 nm laser irradiation, the Ti3C2/Apt-M nanotherapeutic system displayed a satisfactory antitumor effect by targeted photothermal therapy both in vitro and in vivo.ConclusionThis study provides a new idea for the development of MXenes nanotherapeutic system with high active targeting performance.Graphical

Pattern of MUC6 expression across 119 different tumor types: A tissue microarray study on 15 412 tumors.

Mucin 6 (MUC6) is a secreted gel-forming mucin covering the surfaces of gastrointestinal and other tissues. Published work demonstrates that MUC6 can also be expressed in several cancer types and can aid in the distinction of different tumor entities. To systematically analyze MUC6 expression in normal and cancerous tissues, a tissue microarray containing 15 412 samples from 119 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. At least a weak MUC6 positivity was seen in 50 of 119 (42%) tumor entities. Thirty-three tumor entities included tumors with strong positivity. MUC6 immunostaining was most frequent in mucinous carcinomas of the breast (44%), adenocarcinomas of the stomach (30%-40%) and esophagus (35%), and neuroendocrine carcinomas of the colon. Strong MUC6 staining was linked to advanced pT stage (p = 0.0464), defective mismatch repair status and right-sided tumor location (p < 0.0001 each) in colorectal cancer, as well as to high tumor grade (p = 0.0291), nodal metastasis (p = 0.0485), erb-b2 receptor tyrosine kinase 2 positivity (p < 0.0001) and negative estrogen receptor (p = 0.0332)/progesterone receptor (p = 0.0257) status in breast carcinomas of no special type. The broad range of tumor types with MUC6 expression limits the utility of MUC6 immunohistochemistry for the distinction of different tumor types.

Strategies for Synthesizing and Enhancing the Immune Response of Cancer Vaccines Based on MUC1 Glycopeptide Antigens.

Cancer vaccines are based on a vaccinology strategy whereby the patient's immune system is harnessed to induce a specific immune response to kill cancer cells and comprises two categories: prophylactic and therapeutic. Glycoprotein mucin 1 (MUC1), which is overexpressed and poorly glycosylated on cancer cells, is one of the most promising candidates for the development of new cancer vaccines. However, it should be noted that mucin-like glycopeptides are poorly immunogenic and unable to elicit effective and long-lasting immune responses. Therefore, MUC1-derived tumor antigens need to be conjugated with immune activators. This review focuses on the synthesis of MUC1 glycopeptides, provides an overview of recently advanced designs of vaccines based on MUC1, and compares the advantages and disadvantages of the various strategies devised to date.

Caffeic acid improves intestinal barrier functions by regulating colonic bacteria and tight junction protein expression and alleviating inflammation in weaning piglets

The experiment investigated the effect of caffeic acid on bacteria, short-chain fatty acids (SCFA), and the expression of tight junction protein and inflammation related genes in the colon of weaning piglets. Thirty-six weaning piglets were allocated to three treatment groups, which were fed with a basal diet, a basal diet supplemented with 250 mg/kg or 500 mg/kg caffeic acid for 28 days. The results showed that caffeic acid treatment increased the contents of acetate acid, propionate acid and total SCFA. Moreover, real-time quantitative PCR showed that the number of Bifidobacterium (p < 0.05) and Lactobacillus (p < 0.05) were increased and the number of Escherichia coli (p < 0.05) was decreased by caffeic acid in colonic mucosa. Real-time quantitative PCR also showed that the mRNA levels of zonula occludens-1 (p < 0.01), claudin-1 (p < 0.01), occludin (p < 0.01), mucin 1 (MUC1) (p < 0.01), MUC2 (p < 0.01), interleukin 4 (IL-4) (p < 0.01) and IL-10 (p < 0.05) were increased, while the mRNA expression levels of histone deacetylases (p < 0.01), IL-1 (p < 0.01), IL-6 (p < 0.01) and tumor necrosis factor-α (TNF-α) (p < 0.01) were decreased, by caffeic acid in colonic mucosa. These results suggested that caffeic acid could improve intestinal barrier function in weaned pigs, which might be mediated by regulating colonic bacteria and tight junction protein expression and alleviating inflammation.

Interferon-λ3 alleviates intestinal epithelium injury induced by porcine rotavirus in mice

Interferons are a group of glycoproteins that are expressed in various cell types in their inflammatory responses to infections. In this study, we explored the protective effects of porcine interferon-λ3 (PIFN-λ3) on intestinal inflammation and injury in mice induced by porcine rotavirus (PRV). BALB/c mice were administrated by PIFN-λ3 or phosphate buffer solution (PBS) for three days prior to PRV infection. We show that PRV infection caused acute inflammatory responses in mice, as indicated by increases in serum concentrations of inflammatory cytokines such as the interlukin-1β (IL-1β), interlukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (P < 0.05). However, PIFN-λ3 administration not only decreased their concentrations but also elevated the concentrations of immunoglobulin (Ig) M and IgG in the PRV challenged mice (P < 0.05). PRV infection significantly decreased the jejunal villus height and the ratio of villus height to crypt depth (V/C); however, PIFN-λ3 treatment significantly elevated the villus height and the abundance of tight junction protein ZO-1 in the jejunum (P < 0.05). Moreover, PIFN-λ3 decreased the replication of PRV in the jejunal epithelium, but significantly increased the abundance of sIgA and the activities of maltase and sucrase in the PRV-challenged mice (P < 0.05). Interestingly, PIFN-λ3 elevated the expression levels of sodium/glucose cotransporter 1 (SGLT1) and mucin 2 (MUC2) in the PRV-challenged mice (P < 0.05). Moreover, PIFN-λ3 significantly increased the expression levels of IL-10, signal transducer and activator of transcription 1 (STAT1), and critical interferon-stimulated genes such as the 2′-5′ oligoadenylate synthetase-like 1 (OASL1), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and radical S-adenosyl methionine domain containing 2 (RSAD2) in the jejunum upon PRV infection (P < 0.05). The anti-virus and anti-inflammatory effect of PIFN-λ3 should make it an attractive candidate to prevent various pathogen-induced bowel diseases.

Expression and localisation of MUC1 modified with sialylated core-2 O-glycans in mucoepidermoid carcinoma

Mucoepidermoid carcinoma (MEC) is the most frequent of the rare salivary gland malignancies. We previously reported high expression of Mucin 1 (MUC1) modified with sialylated core-2 O-glycans in MEC by using tissue homogenates. In this study, we characterised glycan structures of MEC and identified the localisation of cells expressing these distinctive glycans on MUC1. Mucins were extracted from the frozen tissues of three patients with MEC, and normal salivary glands (NSGs) extracted from seven patients, separated by supported molecular matrix electrophoresis (SMME) and the membranes stained with various lectins. In addition, formalin-fixed, paraffin-embedded sections from three patients with MEC were subjected to immunohistochemistry (IHC) with various monoclonal antibodies and analysed for C2GnT-1 expression by in situ hybridisation (ISH). Lectin blotting of the SMME membranes revealed that glycans on MUC1 from MEC samples contained α2,3-linked sialic acid. In IHC, MUC1 was diffusely detected at MEC-affected regions but was specifically detected at apical membranes in NSGs. ISH showed that C2GnT-1 was expressed at the MUC1-positive in MEC-affected regions but not in the NSG. MEC cells produced MUC1 modified with α2,3-linked sialic acid-containing core-2 O-glycans. MUC1 containing these glycans deserves further study as a new potential diagnostic marker of MEC.

Mucin 4 protein is expressed in B-acute lymphoblastic leukemia and is restricted to BCR::ABL1-positive and BCR::ABL-like subtypes

Mucin 4 (MUC4) is a transmembrane mucin that, like most mucins, is not expressed in normal hematopoietic cells but little is known about its expression in malignant hematopoiesis. B-Acute Lymphoblastic Leukemia (B-ALL) consists of genetically distinct disease subtypes with similarities and differences in gene expression most frequently studied at the mRNA level, which is less amenable to widespread routine clinical use. Here, we demonstrate using immunohistochemistry (IHC) that MUC4 protein is expressed in less than 10% of B-ALL with expression restricted to BCR::ABL1+ and BCR::ABL1-like (CRLF2 rearranged) subtypes of B-ALL (4/13, 31%). None (0/36, 0%) of the remaining B-ALL subtypes expressed MUC4. We compare clinical and pathologic features of MUC4+ and MUC4- BCR::ABL1+/like cases and most significantly report a possible shorter time to relapse for MUC4+ BCR::ABL1 B-ALL that would need to be validated in larger studies. In conclusion, MUC4 is a specific, albeit insensitive, marker for these high-risk subtypes of B-ALL. We propose that MUC4 IHC may be employed diagnostically to rapidly identify these B-ALL subtypes particularly in resource limited settings or when an aspirate sample is not available for ancillary genetic studies.

Blood biomarkers and Ki-67 proliferation marker in breast cancer

Metastasis is the leading cause of death in patients with breast cancer (BC). It is known that the lesion of regional lymph nodes by tumor cells is more common in tumors with higher proliferative activity. Moreover, there is literature evidence on effects of cytokines and proteins upon the migration potential of the tumor. The aim of our work was to study the correlation between the concentrations of cytokines, proteins, and expression of Ki-67 proliferation marker in breast cancer with histology of non-specific invasive carcinoma.On the basis of pathological findings, 16 patients had metastases in regional lymph nodes (group I), and 18 patients had no detectable metastases (group II). Solid-phase enzyme immunoassay was used to determine concentrations of 14 cytokines in the supernatants of immunocompetent blood cells, i.e., IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1β, IL-1ra, TNFα, IFNγ, G-CSF, GM-CSF, VEGF and MCP-1, and concentrations of 6 proteins were determined in blood serum: estrogen and progesterone receptors, cadherin-E (CDH1), plasminogen activator type 1 (PAI-1), mucin 1 (MUC1), heat shock protein 90αA1 (HSP90αA1). Immunohistochemical study of Ki-67 expression was performed in paraffin sections of tumors using monoclonal antibodies.The study showed that Ki-67 expression in tumor tissues and blood concentrations of IL-6, IL-8, IL-1β and TNFα were higher in group I patients. On the contrary, blood concentrations of CDH1 and PAI-1 were higher in group II patients. It was found that Ki-67 showed both inverse correlations with CDH1 and PAI1, and direct correlations with IL-8 and TNFα. CDH1 had a direct correlation with PAI1, and inverse correlations with IL-6, IL-1β and TNFα. The studied cytokines showed direct correlations with each other. The analysis of ROC curves showed good quality and optimal values of the cut-off points for Ki-67 expression, cytokine and protein concentrations, thus allowing best prediction for detectable lymphatic metastasis.On the basis of these results, a quotient was proposed, which represents a ratio of CDH1 contents to the sum of IL-1β and TNFα concentrations in blood samples, which can help identification of the patients with breast cancer at risk for lymphatic metastasis.

Abstract 6325: A novel EGFR x MUC1 bispecific antibody-drug conjugate, BSA01, targets MUC1 transmembrane cleavage products and improves tumor selectivity

Abstract Epidermal growth factor receptor (EGFR) and mucin 1 (MUC1) are tumor-associated antigens that are highly co-expressed in esophageal squamous cell carcinomas, non-small cell lung cancers (NSCLC), and triple negative breast cancers, among others. MUC1, a glycoprotein essential for the formation of the epithelial mucous barrier, is hypoglycosylated and dimerizes with EGFR, a potent oncoprotein, in transformed cells. Dual-targeting of both tumor-associated antigens (TAAs) represents a promising therapeutic strategy to treat common malignancies. We generated a fully human anti-EGFR/MUC1 bispecific antibody-drug conjugate (BSA01) from RenLite® humanized, common light chain mice. BSA01 is conjugated with Monomethyl auristatin E (MMAE). BSA01 targets the extracellular domain of MUC1-C (C-terminal domain of MUC1), which is the membrane-bound MUC1 cleavage product that remains after MUC1 is cleaved, providing more access to the tumor antigen in situ. In addition, we generated affinity and internalization optimized binders targeting EGFR that increases the tumor selectivity, to avoid potential skin toxicity due to ubiquitous basal EGFR expression. Internalization assays demonstrate that the anti-EGFR/MUC1 bispecific antibody used to generate BSA01 was endocytosed in tumor cells co-expressing EGFR/MUC1 more efficiently than mono- or bivalent antibodies targeting EGFR or MUC1 alone. When conjugated with payload, BSA01 also exhibited strong anti-tumor efficacy in vitro against gastric, NSCLC and pancreatic cancer cell lines. Moreover, BSA01 showed similar activity in vivo, effectively inhibiting tumor growth in multiple cell-derived xenograft and patient-derived xenograft models. These results suggest that BSA01 has great clinical potential for treating tumors co-expressing EGFR and MUC1. Citation Format: Yifu Zhang, Chenzhang Shang, Anqi Wang, Jia Zhang, Yuji Liu, Hao Li, Xiaopeng Li, Gao An, Li Hui, Frank An, Yi Yang. A novel EGFR x MUC1 bispecific antibody-drug conjugate, BSA01, targets MUC1 transmembrane cleavage products and improves tumor selectivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6325.

Abstract 451: AICAR targets oncogenic signaling pathways of MUC1-CT

Abstract Lung cancer cells overexpress oncogenic mucin 1 (MUC1), whose C-terminal active subunit (MUC1-CT) is involved in lung tumor formation. Although a specific peptide against MUC1-CT has effectively blocked the MUC1 signaling pathway, intrinsic metabolites targeting MUC1 were not well studied. 5-aminoimidazole-4-carboxamide riboside (AICAR) is a purine biosynthesis intermediate and an analog of adenosine. Our study showed that high AICAR reduced lung tumor cell growth by increasing DNA damage and cell apoptosis. MUC1 was validated as one of the leading AICAR-binding proteins by virtual target screening and thermal stability assay. AICAR treatment dramatically degraded MUC1-CT at protein levels. Whole transcriptome analysis demonstrated that AICAR negatively regulated the Janus kinase (JAK) signaling pathway. Duolink proximity ligation assay revealed that JAK1 interacted with MUC1 directly, and AICAR treatment impaired this protein-protein interaction. Increased epidermal growth factor receptor (EGFR) activity was linked to upregulated expression of MUC1-CT in tissues from an EGFR transgenic mouse lung cancer model. AICAR treatment reduced phosphorylated EGFR, JAK1, and MUC1-CT in lung cancer cells. A mono-treatment with AICAR reduced tumor formation in a mouse lung xenograft model. Combination therapy with AICAR and small-molecule inhibitors against JAK1 or mutant EGFR reduced the growth of lung tissue-derived tumor organoids. Thus, our study has first disclosed a new ligand repressing the MUC1 activity in lung cancer that disrupts protein-protein interactions between MUC1-CT and other oncogenic proteins. Citation Format: Fareesa Aftab, Alice Rodriguez-Fuguet, Luis Silva, Ikei S. Kobayashi, Jiao Sun, Katerina Politi, Elena Levantini, Wei Zhang, Susumu S. Kobayashi, Wen Cai Zhang. AICAR targets oncogenic signaling pathways of MUC1-CT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 451.

Abstract 1779: Fourth-generation chimeric antigen receptor T cells targeting mucin 1 against breast cancer

Abstract Breast cancer (BC) is the most common cause of deaths worldwide, which is required effective treatment strategies. The second-generation chimeric antigen receptor (CAR) T cell therapy was approved by U.S. Food and Drug Administration for hematological malignancies, exhibiting impressive efficacy. However, CAR T cell therapy has not been approved in solid tumors and it has intensively been the subjects of research and development. To develop CAR T cell therapy, it is crucial to identify target antigen specifically expressed on tumor cells. Mucin 1 (MUC1) was previously reported to be overexpressed in several tumors. In this study, we examined MUC1 expression in BC tissues and cultured cell lines, and to improve CAR T cell efficiency, we generated fourth-generation anti-MUC1 CAR T cells (αM.CAR4 T cells), containing anti-MUC1 single-chain variable fragment (scFv), three costimulatory molecules (CD28, CD137, CD27) and CD3ζ. High levels of MUC1 expression were observed in the tissues and cell lines. Comparing to second- and third-generations (αM.CAR2 T and αM.CAR3 T cells), the αM.CAR4 T cells showed greater proliferation in the CAR T cell production process. The anti-tumor functions of all CAR T cells were specific to MUC1 expression since their killing activities were illustrated against MUC1-positive but not MUC1-negative BC cells. Interestingly, at low effector to target ratios, the αM.CAR4 T cells exhibited higher tumor cell lysis than those of αM.CAR2 T and αM.CAR3 T cells. Furthermore, after exposure to MUC1-positive BC cells, the αM.CAR4 T cells expressed lower programmed cell death protein 1 (PD-1) level. Taken together, the αM.CAR4 T cells are potential to be further developed for treatments of MUC1-positive BC and other cancers. Citation Format: Kamonlapat Supimon, Thanich Sangsuwannukul, Mutita Junking, Pa-thai Yenchitsomanus. Fourth-generation chimeric antigen receptor T cells targeting mucin 1 against breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1779.

Chimonanthus nitens Oliv. leaves flavonoids alleviate hyperuricemia by regulating uric acid metabolism and intestinal homeostasis in mice

This study aimed to evaluate the alleviating effect of Chimonanthus nitens Oliv. leaves flavonoids (CLF) on hyperuricemia induced by potassium oxonate in mice. The results showed that CLF lowered the serum levels of uric acid (UA), creatinine and blood urea nitrogen, downregulated hepatic mRNA expressions of xanthine oxidase (XO), phosphate ribose pyrophosphate synthetase (PRPS) and adenosine deaminase (ADA) in hyperuricemia mice. In addition, CLF repaired renal injury by significantly down-regulating mRNA and protein expressions of renal UA reabsorption-related proteins and up-regulating the mRNA and protein expressions of UA secretory-related proteins. Finally, CLF inhibited UA synthesis and promoted UA excretion to alleviate hyperuricemia. Besides, CLF supplementation repaired the intestinal barrier function as demonstrated by significant increased mRNA levels of intestinal zonula occludens-1 (ZO-1), Occludin, mucin 2 (MUC2) and mucin 4 (MUC4), as well as decreased mRNA levels of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in mice. Further research showed that CLF treatment restored intestinal homeostasis mediated by improving the composition of gut microbiota and elevating the abundance of beneficial bacteria like Lactobacillus, Alistipes, Prevotellaceae_UCG-001 and Parasutterella. Overall, our findings revealed a novel function of CLF as a promising therapeutic candidate for the treatment of hyperuricemia.