Madsen, Cathy S.

The HIF‐1 and β‐catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine Mucin 1 (Muc1) protects kidney function and morphology in a mouse model of ischemia‐reperfusion injury (IRI) by stabilizing HIF‐1α, enhancing HIF‐1 downstream signaling and thereby preventing metabolic stress (Pastor‐Soler et al. 2015 Am. J. Physiol. Renal Physiol. 308, F1452‐F1462). We asked if Muc1 regulates the β‐catenin protective pathway during IRI as published data indicate that (i) β‐catenin is found in co‐immunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, (ii) β‐catenin nuclear targeting is MUC1‐dependent in cultured human cells, and (iii) MUC1 prevents β‐catenin phosphorylation by glycogen synthase kinase 3β (GSK3b) and thereby β‐catenin degradation. Using our same mouse model of IRI, and by using immunohistochemistry and immunoblotting of kidney tissue, we found that levels of active GSK3β were significantly lower in kidneys of control mice compared to Muc1 KO mice. Consequently, β‐catenin was significantly increased at 24 and 72 h recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both β‐catenin induction and nuclear targeting were absent in Muc1 KO mice. In accord with these data, we also found downstream induction of β‐catenin pro‐survival factors (activated Akt, survivin, transcription factor TCF4 and its downstream target cyclin D1) and repression of pro‐apoptotic factors (p53, active Bax and cleaved caspase 3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during AKI proceeds by enhancing both the HIF‐1 and β‐catenin protective pathways. Support or Funding Information This work was supported by National Institutes of Health (NIH) Grants DK‐099345 (to T. A. Sutton), DK‐084184 (to N. M. Pastor‐Soler), CA‐64389 (to S. J. Gendler), DK‐62277, DK‐100287, and DK‐095498 (to S. P. Monga), DK‐097889 (to M. M. Al‐bataineh), DK107632, DK‐079307 (Pilot Project) and Samuel and Emma Winters Foundation grant (to R. P. Hughey), and P30‐DK079307 (to the Microscopy Core N. M. Pastor‐Soler and S. Bastacky).

Objectives/HypothesisDevelop a patient‐specific tissue engineered construct for laryngeal reconstruction following a partial laryngectomy.Study DesignBench and animal research.MethodsA construct made from a porous polyethylene scaffold shaped in a canine‐specific configuration and seeded with autologous canine adipose‐derived stem cells in fibrin glue was implanted in a canine following a partial laryngectomy. After 1 year, the construct was first evaluated in vivo with high‐speed imaging and acoustic–aerodynamic measures. It was then explanted and evaluated histologically.ResultsThe canine study at 1 year revealed the construct provided voicing (barking) with acoustic and aerodynamic measures within normal ranges. The canine was able to eat and breathe normally without long‐term support. The construct was integrated with epithelialization of all areas except the medial portion of the vocal fold structure. No anti‐infective agents were needed after the standard perioperative medications were completed.ConclusionThis study provided a successful first step toward developing a patient‐specific composite construct for patients undergoing partial laryngectomies.Level of EvidenceNA Laryngoscope, 132:S1–S11, 2022

Inositol hexaphosphate (IP6 or phytic acid) is thought to be the active ingredient in legumes and cereals that provides anti‐cancer activity. Our goal is to understand the influence of IP6 on mucin expression (Muc1 and Muc2, specifically) in the mouse intestine. C57BL/6 mice were randomized into 3 groups of 6 mice. Mice were orally dosed with IP6 (0, 1, 2 g/kg BW) for 5 days and sacrificed on the sixth day. Sections of the small intestine were removed, fixed in methacarn, sectioned and stained with Alcian blue‐PAS to visualize mucins. Mucosa from the jejunum and colon were removed and total RNA prepared using Trizol®. cDNA was prepared using random hexamers and TaqMan minor groove binding probes were used for real time PCR amplification of message for Muc1 and Muc2. Expression of Muc1 in the jejunum was minimal whereas that of Muc2 increased 1.4‐fold at the higher IP6 dose rate. In the colon, expression of both Muc1 and Muc2 increased in mice dosed with IP6. Expression of Muc1 increased 1.6‐fold and 3.8‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Expression of Muc2 increased 2.0‐fold and 1.7‐fold in mice dosed with 1 and 2 g/kg IP6, respectively. Alcian blue‐PAS stained sections of the colon showed an increase in mucus. Thus, IP6 seems to increase the expression of Muc1 and Muc2 in the mucosa lining of the small intestine, particularly in the colon. Research was supported by East Tennessee State University and Mayo Clinic.

Background Though the importance of the transmembrane mucin MUC1 in mammary oncogenesis has long been recognized, the relative contributions of the cytoplasmic tail and tandem repeat domains are poorly understood. Methods To address this, mouse models of mammary carcinogenesis were created expressing full-length cytoplasmic tail-deleted, or tandem repeat-deleted MUC1 constructs. Results Overexpression of full-length MUC1 resulted in tumor formation in young mice (≤ 12 months); however, loss of either the cytoplasmic tail or the tandem repeat domain abrogated this oncogenic capacity. Aged mice in all strains developed late-onset mammary tumors similar to those previously described for the FVB background. Conclusions This study is the first spontaneous cancer model to address the relative importance of the cytoplasmic tail and tandem repeat domains to MUC1-driven mammary oncogenesis, and suggests that both of these domains are essential for tumor formation.

AbstractA fully synthetic MUC1‐based cancer vaccine was designed and chemically synthesized containing an endogenous helper T‐epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody‐dependent cell‐mediated cytotoxicity (ADCC). It also activated cytotoxic T‐lymphocytes. Collectively, the immunological data demonstrate engagement of helper T‐cells in immune activation. A synthetic methodology was developed for a penta‐glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1‐based cancer vaccines that elicited potent immune responses employed exogenous helper T‐epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T‐epitope will be more attractive, because it will activate cognate CD4+ T‐cells that will provide critical tumor‐specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.

The hypoxia-inducible factor (HIF)-1 and β-catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine mucin (Muc)1 protects kidney function and morphology in a mouse model of ischemia-reperfusion injury (IRI) by stabilizing HIF-1α, enhancing HIF-1 downstream signaling, and thereby preventing metabolic stress (Pastor-Soler et al. Muc1 is protective during kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol 308: F1452–F1462, 2015). We asked if Muc1 regulates the β-catenin protective pathway during IRI as 1) β-catenin nuclear targeting is MUC1 dependent in cultured human cells, 2) β-catenin is found in coimmunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, and 3) MUC1 prevents β-catenin phosphorylation by glycogen synthase kinase (GSK)3β and thereby β-catenin degradation. Using the same mouse model of IRI, we found that levels of active GSK3β were significantly lower in kidneys of control mice compared with Muc1 knockout (KO) mice. Consequently, β-catenin was significantly upregulated at 24 and 72 h of recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both β-catenin induction and nuclear targeting were absent in Muc1 KO mice. We also found downstream induction of β-catenin prosurvival factors (activated Akt, survivin, transcription factor T cell factor 4 (TCF4), and its downstream target cyclin D1) and repression of proapoptotic factors (p53, active Bax, and cleaved caspase-3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during acute kidney injury proceeds by enhancing both the HIF-1 and β-catenin protective pathways.

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells.

A cystic fibrosis (CF) mouse expressing the human mucin MUC1 transgene (CFM) reverted the CF/Muc1−/−phenotype (little mucus accumulated in the intestine) to that of CF mice expressing mouse Muc1, which exhibited increased mucus accumulation. Western blots and immunohistochemical analysis showed that the MUC1 protein was markedly increased in CFM mice in which it was both membrane bound and secreted into the intestinal lumen. Studies to determine the reason for increased levels of the extracellular domain of MUC1 mucin identified mRNA and protein of two novel splice variants and the previously described secreted MUC1 lacking the cytoplasmic tail (MUC1/SEC). Novel MUC1 splice variants, CT80 and CT58, were both transmembrane proteins with cytoplasmic tails different from the normal MUC1. The MUC1-CT80 and MUC1/SEC forms are found expressed mainly in the CFM mice intestines. Thus MUC1 expression is increased, and it appears that alternate cytoplasmic tails may change its role in signaling. MUC1 could be an important contributor to the CF intestinal phenotype.

Abstract Pancreatic cancer is a highly aggressive, treatment refractory cancer and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas overexpress altered forms of a tumor-specific Ag, mucin 1 (MUC1; an epithelial mucin glycoprotein), which is a potential target for immunotherapy. We have established a clinically relevant animal model for pancreatic cancer by developing a double transgenic mouse model (called MET) that expresses human MUC1 as self molecule and develops spontaneous tumors of the pancreas. These mice exhibit acinar cell dysplasia at birth, which progresses to microadenomas and acinar cell carcinomas. The tumors express large amounts of underglycosylated MUC1 similar to humans. Tumor-bearing MET mice develop low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies.

Abstract Purpose: MUC1 is a tumor-associated antigen that is aberrantly expressed in cancer and inflammatory bowel disease (IBD). Even though immune cells express low MUC1 levels, their modulations of MUC1 are important in tumor progression. Consistent with previous clinical data that show increased myeloid-derived suppressor cells (MDSCs) in IBD, we now show that downregulation of MUC1 on hematopoietic cells increases MDSCs in IBD, similar to our data in tumor-bearing mice. We hypothesize that MDSC expansion in IBD is critical for tumor progression. Experimental Design: To mechanistically confirm the linkage between Muc1 downregulation and MDSC expansion, we generated chimeric mice that did not express Muc1 in the hematopoietic compartment (KO→WT). These mice were used in two models of colitis and colitis-associated cancer (CAC) and their responses were compared with wild-type (WT) chimeras (WT→WT). Results: KO→WT mice show increased levels of MDSCs during colitis and increased protumorigenic signaling in the colon during CAC, resulting in larger colon tumors. RNA and protein analysis show increased upregulation of metalloproteinases, collagenases, defensins, complements, growth factors, cytokines, and chemokines in KO→WT mice as compared with WT→WT mice. Antibody-mediated depletion of MDSCs in mice during colitis reduced colon tumor formation during CAC. Conclusion: Development of CAC is a serious complication of colitis and our data highlight MDSCs as a targetable link between inflammation and cancer. In addition, the lack of MUC1 expression on MDSCs can be a novel marker for MDSCs, given that MDSCs are still not well characterized in human cancers. Clin Cancer Res; 19(18); 5039–52. ©2013 AACR.