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MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant rMUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Little is known about the pro-resolution role of immune cells recruited to damaged tissue. Using an experimental model of intestinal epithelial damage and repair, we identified B cells as the dominant cell type in the healing colon. Single-cell RNA-sequencing (scRNAseq) revealed the expansion of an IFN-induced B cell subset during experimental mucosal healing which was associated with colitis severity. In line with this, B cell depletion during mucosal healing resulted in accelerated recovery upon injury, which was associated with enhanced expression of tissue remodeling genes. scRNA-seq from the epithelial and stromal compartment confirmed that lack of B cells during mucosal healing alters gene activity programs associated with tissue remodeling. Combined scRNAseq and spatial transcriptomic analysis showed that IFN-induced B cells are located at sites of damage/remodeling and that absence of B cells resulted in decreased potential interaction and co-localization between stromal and epithelial cells. Thus, we identified a previously undescribed role of B cells impairing cell-cell interactions during mucosal healing.

Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X over-expression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-β1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC.

The aryl hydrocarbon receptor (AHR) controls interleukin 22 production by T helper 17 cells (Th17). IL-22 contributes to intestinal homeostasis but has also been implicated in chronic inflammatory disorders and colorectal cancer, highlighting the need for appropriate regulation of IL-22 production. Upon activation, the AHR induces expression of cytochrome P4501 (CYP1) enzymes which in turn play an important feedback role that curtails the duration of AHR signaling by metabolizing AHR ligands. Recently we described how agents that inhibit CYP1 function potentiate AHR signaling by disrupting metabolic clearance of the endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ). In the present study, we investigated the immune-modulating effects of environmental pollutants such as polycyclic aromatic hydrocarbons on Th17 differentiation and IL-22 production. Using Th17 cells deficient in CYP1 enzymes (Cyp1a1/1a2/1b1-/-) we show that these chemicals potentiate AHR activation through inhibition of CYP1 enzymes which leads to increases in intracellular AHR agonists. Our findings demonstrate that IL-22 production by Th17 cells is profoundly enhanced by impaired CYP1-function and strongly suggest that chemicals able to modify CYP1 function or expression may disrupt AHR-mediated immune regulation by altering the levels of endogenous AHR agonist(s).

The gastrointestinal microenvironment, dominated by dietary compounds and the commensal bacteria, is a major driver of intestinal CD4+ T helper (Th) cell differentiation. Dietary compounds can be sensed by nuclear receptors (NRs) that consequently exerts pleiotropic effects including immune modulation. However, how NRs regulate distinct intestinal Th subsets remain poorly understood. Here, we found that under homeostatic condition Liver X receptor (LXR), a sensor of cholesterol metabolites, controls RORγt+ Treg and Th17 cells in the intestine draining mesenteric lymph node (MLN). Mechanistically, while lack of LXR signaling in CD11c+ myeloid cells led to an increase in RORγt+ Treg, modulation of MLN Th17 was independent of LXR signaling in either immune or epithelial cells. Of note, LXRα modulated only the Th17 cells, but not RORγt+ Treg in the MLN and horizontal transfer of microbiota between LXRα−/− and WT mice was sufficient to partially increase the MLN Th17 in WT mice. While LXRα deficiency increased the abundance of Ruminococcaceae and Lachnospiraceae bacterial families compared to the WT littermates, microbiota ablation including ablation of SFB was not sufficient to dampen LXRα-mediated expansion of MLN Th17. Altogether, our results suggest that LXR modulates RORγt+ Treg and Th17 cells in the MLN through distinct mechanisms.

Nuclear receptors (NRs) rapidly activate/repress gene expression to detour immune responses and allow tissue adaptation to constant environmental changes. However, the effect of combined NRs in the immune system is often unclear due to the lack of reliable experimental models that recapitulate the complex interaction between NRs in vivo. Here, we used the zebrafish to investigate the immunological outcome of combining the activation of retinoic acid receptor (RAR), liver X receptor (LXR) and the cytoplasmic sensor aryl hydrocarbon receptor (AHR). Although simultaneous activation did not affect the expression of respective bona-fide target genes, RAR-induced il17a/f3 was antagonized by LXR and AHR, whereas il22 was antagonized by AHR but not LXR. In addition, RA decreased il10 expression, which was further decreased by LXR activation. Thus, using combinatorial NR activation in zebrafish larvae, we show that LXR antagonizes the expression of selected RA-induced cytokines and provide a strategy to tailor the cytokine milieu.

The intestinal environment harbors a large number of activated T cells, which are potentially inflammatory. To prevent inflammatory responses, intestinal T cells are controlled by various tolerogenic mechanisms, including T cell apoptosis. We investigated the expression mechanism and function of the purinergic receptor P2X7 in contraction of intestinal CD4+ effector T cells. We found that P2X7 up-regulation on CD4+ effector T cells is induced by retinoic acid through retinoic acid receptor α (RARα) binding to an intragenic enhancer region of the P2rx7 gene. P2X7 is highly expressed by most intestinal αβ and γδ T cells, including Th1 and Th17 cells. The intestinal effector T cells are effectively deleted by P2X7 activation-dependent apoptosis. Moreover, P2X7 activation suppressed T cell-induced colitis in Rag1−/− mice. The data from vitamin A-deficient and P2rx7−/− mice indicate that the retinoic acid-P2X7 pathway is important in preventing aberrant build-up of activated T cells. We conclude that retinoic acid controls intestinal effector T cell populations by inducing P2X7 expression. These findings have important ramifications in preventing inflammatory diseases in the intestine.

MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.

MUC4, a large transmembrane mucin normally expressed in the small and large intestine, is differentially expressed during inflammatory and malignant conditions of the colon. However, the expression pattern and the role of MUC4 in colitis and colorectal cancer (CRC) are inconclusive. Therefore, the aim of this study was to understand the role of Muc4 during inflammatory and malignant conditions of the colon. Here, we generated Muc4(-/-) mice and addressed its role in colitis and colitis-associated CRC using dextran sodium sulfate (DSS) and azoxymethane (AOM)-DSS experimental models, respectively. Muc4(-/-) mice were viable, fertile with no apparent defects. Muc4(-/-) mice displayed increased resistance to DSS-induced colitis compared with wild-type (WT) littermates that was evaluated by survival rate, body weight loss, diarrhea and fecal blood score, and histological score. Reduced infiltration of inflammatory cells, that is, CD3(+) lymphocytes and F4/80(+) macrophages was observed in the inflamed mucosa along with reduction in the mRNA levels of inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α and anti-microbial genes Lysozyme M and SLPI in the colon of Muc4(-/-) mice compared with WT littermates. Compensatory upregulation of Muc2 and Muc3 mucins under basal and DSS treatment conditions partly explains the resistance observed in Muc4(-/-) mice. Accordingly, Muc4(-/-) mice exhibited significantly reduced tumor burden compared with WT mice assessed in a colitis-induced tumor model using AOM/DSS. An increased percentage of Ki67(+) nuclei was observed in the tumors from WT compared with Muc4(-/-) mice suggesting Muc4 to be critical in intestinal cell proliferation during tumorigenesis. Taken together, we conclusively demonstrate for the first time the role of Muc4 in driving intestinal inflammation and inflammation-associated tumorigenesis using a novel Muc4(-/-) mouse model.

Pancreatic cancer (PC) is characterized by aberrant overexpression of mucins that contribute to its pathogenesis. Although the inflammatory cytokines contribute to mucin overexpression, the mucin profile of PC is markedly distinct from that of normal or inflamed pancreas. We postulated that de novo expression of various mucins in PC involves chromatin modifications. Analysis of chromatin modifying enzymes by PCR array identified differential expression of NCOA3 in MUC4-expressing PC cell lines. Immunohistochemistry analysis in tumor tissues from patients and spontaneous mouse models, and microarray analysis following the knockdown of NCOA3 were performed to elucidate its role in mucin regulation and overall impact on PC. Silencing of NCOA3 in PC cell lines resulted in significant downregulation of two most differentially expressed mucins in PC, MUC4 and MUC1 (P

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of retinoic acid receptor alpha (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack or RARα resulted in increased KLF4+ goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased reg3g, reduced luminal bacterial detection and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

Vitamin A (VA) is amongst the most well characterized food-derived nutrients with diverse immune modulatory roles. Deficiency in dietary VA has not only been associated with immune dysfunctions in the gut, but also with several systemic immune disorders. In particular, VA metabolite all-trans retinoic acid (atRA) has been shown to be crucial in inducing gut tropism in lymphocytes and modulating T helper differentiation. In addition to the widely recognized role in adaptive immunity, increasing evidence identifies atRA as an important modulator of innate immune cells, such as tolerogenic dendritic cells (DCs) and innate lymphoid cells (ILCs). Here, we focus on the role of retinoic acid in differentiation, trafficking and the functions of innate immune cells in health and inflammation associated disorders. Lastly, we discuss the potential involvement of atRA during the plausible crosstalk between DCs and ILCs.

Pancreatic cancer is one of the most lethal cancers in the world, as it continues to be resistant to any therapeutic approaches. The high molecular weight glycoprotein mucin 4 (MUC4) is aberrantly expressed in pancreatic cancer and contributes to the regulation of differentiation, proliferation, metastasis, and the chemoresistance of pancreatic cancer cells. The absence of its expression in the normal pancreatic ductal cells makes MUC4 a promising target for novel cancer therapeutics. Natural products have been widely investigated as potential candidates in cancer therapies, and thymoquinone (TQ), extracted from the seeds of Nigella sativa, has shown excellent antineoplastic properties in some systems. In the present study, we evaluated the effect of TQ on pancreatic cancer cells and specifically investigated its effect on MUC4 expression. The MUC4-expressing pancreatic cancer cells FG/COLO357 and CD18/HPAF were incubated with TQ, and in vitro functional assays were done. The results obtained indicate that treatment with TQ downregulated MUC4 expression through the proteasomal pathway and induced apoptosis in pancreatic cancer cells by the activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase pathways. In agreement with previous studies, the decrease in MUC4 expression correlated with an increase in apoptosis, decreased motility, and decreased migration of pancreatic cancer cells. MUC4 transient silencing studies showed that c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase pathways are activated in pancreatic cancer cells, indicating that the activation of these pathways by TQ is directly related to the MUC4 downregulation induced by the drug. Overall, TQ has potential for the development of novel therapies against pancreatic cancer. Mol Cancer Ther; 9(5); 1419–31. ©2010 AACR.

MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is upregulated in multiple malignancies and is associated with poor prognosis. While thepro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associatedcarboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavagegenerating MUC16-Cter has remained unknown. Using different lengths of dual-epitope(N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, wedemonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretchof twelve amino acids that generates a ~17 kDa cleaved product and isdistinct from the predicted sites. This was further corroborated by domain swappingexperiment. Further, the cleavage of MUC16 was found to take place in theGolgi/post-Golgi compartments and is dependent on the acidic pH in the secretorypathway. A similar pattern of ~17 kDa cleaved MUC16 was observed inmultiple cell types eliminating the possibility of cell type specific phenomenon.MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds tothe chromatin suggesting its involvement in regulation of gene expression. Takentogether, we demonstrate for the first time the oft-predicted cleavage of MUC16 thatis critical in designing successful therapeutic interventions based on MUC16.

Mucins are high molecular weight glycoproteins that are involved in regulating diverse cellular activities both in normal and pathological conditions. Mucin activity and localization is mediated by several molecular mechanisms, including discrete interactions with other proteins. An understanding of the biochemistry behind the known interactions between mucins and other proteins, coupled with an appreciation of their pathophysiological significance, can lend insight into the development of novel therapeutic agents. Indeed, a recent study demonstrated that a cell permeable inhibitor, PMIP, that disrupts the MUC1–EGFR interaction, is effective in killing breast cancer cells in vitro and in tumor models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC MUC16, also known as CA125, is routinely used to monitor the progression (as a biomarker) and response to therapeutics (as a prognostic marker) for epithelial ovarian cancer (EOC), but the deregulated expression is not restricted only to EOC. Recent studies from our lab have shown that normal breast and pancreas (ducts) do not express MUC16; however its expression progressively increases in primary and metastatic breast and pancreatic cancers. In addition, we have observed the involvement of MUC16 in enhancing the tumorigenic potential by shRNA studies. In the present study, to understand the mechanistic involvement of MUC16, we have focused on its C-terminal region. MUC16 is a type-I membrane-bound mucin with a heavily glycosylated N-terminal domain, a transmembrane region (TM) and a 32-residue cytoplasmic tail (CT) domain. Though it has been predicted that MUC16 undergoes cleavage/shedding in the extracellular SEA domain(s) by proteases such as MMP7 and neutrophil elastase, the exact nature and site of cleavage and the fate of the membrane bound portion (C-terminal) after release of extracellular portion is not entirely understood. The post cleavage C-ter region is interesting due to presence of (a) stretch of polybasic aminoacids, the site of interaction for cytoskeletal proteins like Ezrin/Radixin/Moesin, (b) potential nuclear localization signal (bioinformatics prediction) which might facilitate its nuclear localization with associated transcription factors modulating the transcription of various target genes and (c) several potential serine, threonine and tyrosine phosphorylation sites facilitating its interaction(s) with proteins influencing oncogenic signaling pathways. Therefore, we hypothesized that the oncogenic potential of MUC16 is in part, mediated by the potential involvement of C-terminal domain of MUC16. Thusly, we have made several truncated versions of MUC16 from the C-terminal end with N-terminal FLAG tag and C-terminal HA-tag and are in the process of characterization. To understand the functional significance of this C-terminal region, we have stably transfected MCF7 (breast cancer), MiaPaCa and T3M4 (pancreatic cancer) cells with an empty vector, FLAG-MUC16-114AA-HA and FLAG-MUC16-114AA-dCT (32 AA in the CT is deleted). Our results from in vitro functional assays indicate that C-terminal region of MUC16 is responsible for enhancing the invasive potential of cancer cells and confers resistance to chemotherapeutic agents such as Cisplatin and Gemcitabine. Moreover, we see that the function of the carboxyl terminal MUC16 is dependent on the stability of the protein, which is a function of N-glycosylation and ubiquitylation. Overall, our studies indicate that C-terminal region of MUC16 is an important mediator of cancer cell invasion and drug resistance. (This work is supported from the Department of Defense [BC101014][1]). Citation Format: Srustidhar Das, Maria P. Torres-Gonzalez, Prabin D. Majhi, Imayavaramban Lakshmanan, Moorthy P. Ponnusamy, Satyanarayana Rachagani, Eric Cruz, Dhanya Haridas, Surinder K. Batra. Carboxyl terminal region of MUC16 is critical for tumor cell invasion and drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5113. doi:10.1158/1538-7445.AM2013-5113 [1]: /lookup/external-ref?link_type=GEN&access_num=BC101014&atom=%2Fcanres%2F73%2F8_Supplement%2F5113.atom