Molecular crosstalk between MUC1 and STAT3 influences the anti-proliferative effect of Napabucasin in epithelial cancers.

MUC1 is a transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in epithelial cancers. The cytoplasmic tail of MUC1 (MUC1 CT) aids in tumorigenesis by upregulating the expression of multiple oncogenes. Signal transducer and activator of transcription 3 (STAT3) plays a crucial role in several cellular processes and is aberrantly activated in many cancers. In this study, we focus on recent evidence suggesting that STAT3 and MUC1 regulate each other's expression in cancer cells in an auto-inductive loop and found that their interaction plays a prominent role in mediating epithelial-to-mesenchymal transition (EMT) and drug resistance. The STAT3 inhibitor Napabucasin was in clinical trials but was discontinued due to futility. We found that higher expression of MUC1 increased the sensitivity of cancer cells to Napabucasin. Therefore, high-MUC1 tumors may have a better outcome to Napabucasin therapy. We report how MUC1 regulates STAT3 activity and provide a new perspective on repurposing the STAT3-inhibitor Napabucasin to improve clinical outcome of epithelial cancer treatment.

Mucin-1-Targeted Chimeric Antigen Receptor T Cells Are Effective and Safe in Controlling Solid Tumors in Immunocompetent Host.

The chimeric antigen receptor (CAR) T-cell therapy in solid epithelial tumors has been explored, however, with limited success. As much of the preclinical work has relied on xenograft models in immunocompromised animals, the immune-related efficacies and toxicities may have been missed. In this study, we engineered syngeneic murine CAR T cells targeting the tumor form of human mucin-1 (tMUC1) and tested the MUC1 CAR T cells' efficacy and toxicity in the immunocompetent human MUC1-expressing mouse models. The MUC1 CAR T cells significantly eliminated murine pancreatic and breast cancer cell lines in vitro. In vivo, MUC1 CAR T cells significantly slowed the mammary gland tumor progression in the spontaneous PyVMT×MUC1.Tg (MMT) mice, prevented lung metastasis, and prolonged survival. Most importantly, there was minimal short or long-term toxicity with acceptable levels of transient liver toxicity but no kidney toxicity. In addition, the mice did not show any signs of weight loss or other behavioral changes with the treatment. We also report that a single dose of MUC1 CAR T-cell treatment modestly reduced the pancreatic tumor burden in a syngeneic orthotopic model of pancreatic ductal adenocarcinoma given at late stage of an established tumor. Taken together, these findings suggested the further development of tMUC1-targeted CAR T cells as an effective and relatively safe treatment modality for various tMUC1-expressing solid tumors.

Targeting tumor-associated MUC1 overcomes anoikis-resistance in pancreatic cancer.

The third leading cause of cancer-related deaths in the United States is pancreatic cancer, more than 95% of which is pancreatic ductal adenocarcinoma (PDA). The incidence rate of PDA nearly matches its mortality rate and the best treatment till date is surgical resection for which only 25% are eligible. Tumor recurrence and metastasis are the main causes of cancer-related mortality. MUC1 is a transmembrane glycoprotein expressed on most epithelial cells. It is overexpressed and aberrantly glycosylated in cancer and is known as tumor-associated MUC1 (tMUC1). More than 80% of PDAs express tMUC1. A monoclonal antibody called TAB004 has been developed specifically against human tMUC1 extracellular domain. We report that treatment with TAB004 significantly reduced the colony forming potential of multiple PDA cell lines while sparing normal pancreatic epithelial cell line. Binding of TAB004 to tMUC1 compromised desmosomal integrity, induced ER stress and anoikis in PDA cells. The mechanisms underlying TAB004's antitumor effects were found to be reduced activation of the EGFR-PI3K signaling pathway, and degradation of tMUC1, thereby reducing expression of its transcriptional targets, c-Src and c-Myc. This reduction in oncogenic signaling triggered anoikis as indicated by reduced expression of antiapoptotic proteins, PTRH2 and BCL2. TAB004 treatment slowed the growth of PDA xenograft compared to IgG control and enhanced survival of mice when combined with 5-FU. Since TAB004 significantly reduced colony forming potential and triggered anoikis in the PDA cells, we suggest that it could be used as a potential prophylactic agent to curb tumor relapse after surgery, prevent metastasis and help increase the efficacy of chemotherapeutic agents.

Nanoparticle combination for precise stroma modulation and improved delivery for pancreatic cancer.

Therapeutic success in the treatment of pancreatic ductal adenocarcinoma (PDAC) is hindered by the extensive stroma associated to this disease. Stroma is composed of cellular and non-cellular components supporting and evolving with the tumor. One of the most studied mediators of cancer cell-stroma crosstalk is sonic hedgehog (SHh) pathway leading to the intense desmoplasia observed in PDAC tumors. Herein, we demonstrate that the use of mesoporous silica nanoparticles (MSNs) containing an SHh inhibitor, cyclopamine (CyP), and the combination of chemotherapeutic drugs (Gemcitabine (Gem)/cisplatin (cisPt)) as the main delivery system for the sequential treatment led to the reduction in tumor stroma along with an improvement in the treatment of PDAC. We synthesized two versions of the MSN-based platform containing the SHh inhibitor (CyP-MSNs) and the drug combination (PEG-Gem-cisPt-MSNs). In vitro and in vivo protein analysis show that CyP-MSNs effectively inhibited the SHh pathway. In addition, the sequential combination of CyP-MSNs followed by PEG-Gem-cisPt-MSNs led to effective stromal modulation, increased access of secondary PEG-Gem-cisPt-MSNs at the tumor site, and improved therapeutic performance in HPAF II xenograft mice. Taken together, our findings support the potential of drug delivery using MSNs for stroma modulation and to prevent pancreatic cancer progression.

Overexpression of MUC1 Induces Non-Canonical TGF-ß Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human cancers. Transforming Growth Factor Beta (TGF-ß) is a cytokine that switches from a tumor-suppressor at early stages to a tumor promoter in the late stages of tumor development, by yet unknown mechanisms. Tumor associated MUC1 is aberrantly glycosylated and overexpressed in >80% of PDAs and is associated with poor prognosis. MUC1 expression is found in the early stages of PDA development with subsequent increase in later stages. Analysis of human PDA samples from TCGA database showed significant differences in gene expression and survival profiles between low and high MUC1 samples. Further, high MUC1 expression was found to positively correlate to TGF-ßRII expression and negatively correlate to TGF-ßRI expression in PDA cell lines. We hypothesized that MUC1 overexpression induces TGF-ß mediated non-canonical signaling pathways which is known to be associated with poor prognosis. In this study, we report that MUC1 overexpression in PDA cells directly activates the JNK pathway in response to TGF-ß, and leads to increased cell viability via up-regulation and stabilization of c-Myc. Conversely, in low MUC1 expressing PDA cells, TGF-ß preserves its tumor-suppressive function and inhibits phosphorylation of JNK and stabilization of c-Myc. Knockdown of MUC1 in PDA cells also results in decreased phosphorylation of JNK and c-Myc in response to TGF-ß treatment. Taken together, the results indicate that overexpression of MUC1 plays a significant role in switching the TGF-ß function from a tumor-suppressor to a tumor promoter by directly activating JNK. Lastly, we report that high-MUC1 PDA tumors respond to TGF-ß neutralizing antibody in vivo showing significantly reduced tumor growth while low-MUC1 tumors do not respond to TGF-ß neutralizing antibody further confirming our hypothesis.

Advanced Nanoengineering Approach for Target-Specific, Spatiotemporal, and Ratiometric Delivery of Gemcitabine-Cisplatin Combination for Improved Therapeutic Outcome in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an intractable malignancy with a dismal survival rate. Recent combination therapies have had a major impact on the improvement of PDAC prognosis. Nevertheless, clinically used combination regimens such as FOLFIRINOX and gemcitabine (Gem)/nab-paclitaxel still face major challenges due to lack of the safe and ratiometric delivery of multiple drugs. Here, a rationally designed mesoporous silica nanoparticle (MSN)-based platform is reported for the target-specific, spatiotemporal, ratiometric, and safe co-delivery of Gem and cisplatin (cisPt). It is shown that systemic administration of the nanoparticles results in synergistic therapeutic outcome in a syngeneic and clinically relevant genetically engineered PDAC mouse model that has rarely been used for the therapeutic evaluation of nanomedicine. This synergism is associated with a strategic engineering approach, in which nanoparticles provide redox-responsive controlled delivery and in situ differential release of Gem/cisPt drugs with the goal of overcoming resistance to Pt-based drugs. The platform is also rendered with additional tumor-specificity via a novel tumor-associated mucin1 (tMUC1)-specific antibody, TAB004. Overall, the platform suppresses tumor growth and eliminates the off-target toxicities of a highly toxic chemotherapy combination.

Repression of MUC1 Promotes Expansion and Suppressive Function of Myeloid-Derived Suppressor Cells in Pancreatic and Breast Cancer Murine Models.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing syngeneic pancreatic (KCKO) or breast (C57MG) tumors. We observed enhanced tumor growth of pancreatic and breast tumors in the MUC1KO mice compared to the WT mice. Enhanced tumor growth in the MUC1KO mice was associated with increased numbers of suppressive MDSCs and T regulatory (Tregs) cells in the tumor microenvironment. Compared to the WT host, MUC1KO host showed higher levels of iNOS, ARG1, and TGF-ß, thus promoting proliferation of MDSCs with an immature and immune suppressive phenotype. When co-cultured with effector T cells, MDSCs from MUC1KO mice led to higher repression of IL-2 and IFN-γ production by T cells as compared to MDSCs from WT mice. Lastly, MDSCs from MUC1KO mice showed higher levels of c-Myc and activated pSTAT3 as compared to MDSCs from WT mice, suggesting increased survival, proliferation, and prevention of maturation of MDSCs in the MUC1KO host. We report diminished T cell function in the KO versus WT mice. In summary, the data suggest that MUC1 may regulate signaling pathways that are critical to maintain the immunosuppressive properties of MDSCs.

Reply to the letter from Dr. Pirjo Pärnänen et al.

Nutraceuticals could be used to increase immunity against viral infections and therefore more research should be done on them to better manage COVID-19 and future pandemics.

Mucin signature as a potential tool to predict susceptibility to COVID-19.

The Corona Virus Infectious Disease-19 (COVID-19) pandemic has played havoc on both the global health and economy. It is necessary to find a molecular signature that differentiates between low-risk and high-risk individuals. Pathogens, including viruses of the upper respiratory tract, utilize mucin proteins to enter into host cells. Mucins are critical components of innate immunity and also play important roles in infectious disease progression. Their expression is regulated by different cytokines during infection and inflammation. A comparison of mucin signatures between an asymptomatic versus symptomatic and between patients with mild versus severe symptoms could help identify other important proteins involved in the pathology of this new virus. Recent studies on the pathogenicity of the SARS-CoV-2 have found receptors that help its entry into the cells. In this review, we present an overview of how mucins are connected to the pathogenicity of the virus and propose that studying the glycome and mucin signature may lead to the development of a biomarker in predicting the susceptibility, progression, and response to therapy in COVID-19 patients.

Corrigendum: CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth.

[This corrects the article DOI: 10.3389/fimmu.2019.01149.].

Potential of Anti-MUC1 Antibodies as a Targeted Therapy for Gastrointestinal Cancers.

Gastrointestinal cancers (GI) account for 26% of cancer incidences globally and 35% of all cancer-related deaths. The main challenge is to target cancer specific antigens. Mucins are heavily O-glycosylated proteins overexpressed in different cancers. The transmembrane glycoprotein MUC1 is the most likeable target for antibodies, owing to its specific overexpression and aberrant glycosylation in many types of cancers. For the past 30 years, MUC1 has remained a possible diagnostic marker and therapeutic target. Despite initiation of numerous clinical trials, a comprehensively effective therapy with clinical benefit is yet to be achieved. However, the interest in MUC1 as a therapeutic target remains unaltered. For all translational studies, it is important to incorporate updated relevant research findings into therapeutic strategies. In this review we present an overview of the antibodies targeting MUC1 in GI cancers, their potential role in immunotherapy (i.e., antibody-drug and radioimmunoconjugates, CAR-T cells), and other novel therapeutic strategies. We also present our perspectives on how the mechanisms of action of different anti-MUC1 antibodies can target specific hallmarks of cancer and therefore be utilized as a combination therapy for better clinical outcomes.

Preclinical evaluation of an 111In/225Ac theranostic targeting transformed MUC1 for triple negative breast cancer.

Rationale: Transformed MUC1 (tMUC1) is a cancer-associated antigen that is overexpressed in >90% of triple-negative breast cancers (TNBC), a highly metastatic and aggressive subtype of breast cancer. TAB004, a murine antibody targeting tMUC1, has shown efficacy for the targeted delivery of therapeutics to cancer cells. Our aim was to evaluate humanized TAB004 (hTAB004) as a potential theranostic for TNBC. Methods: The internalization of hTAB004 in tMUC1 expressing HCC70 cells was assessed via fluorescent microscopy. hTAB004 was DOTA-conjugated and radiolabeled with Indium-111 or Actinium-225 and tested for stability and tMUC1 binding (ELISA, flow cytometry). Lastly, in vivo biodistribution (SPECT-CT), dosimetry, and efficacy of hTAB004 were evaluated using a TNBC orthotopic mouse model. Results: hTAB004 was shown to bind and internalize into tMUC1-expressing cells. A production method of 225Ac-DOTA-hTAB004 (yield>97%, RCP>97% SA=5 kBq/µg) and 111In-DOTA-hTAB004 (yield>70%, RCP>99%, SA=884 kBq/µg) was developed. The labeled molecules retained their affinity to tMUC1 and were stable in formulation and mouse serum. In NSG female mice bearing orthotopic HCC70 xenografts, the in vivo tumor concentration of 111In-DOTA-hTAB004 was 65 ± 15 %ID/g (120 h post injection). A single 225Ac-DOTA-hTAB004 dose (18.5 kBq) caused a significant reduction in tumor volume (P<0.001, day 22) and increased survival compared to controls (P<0.007). The human dosimetry results were comparable to other clinically used agents. Conclusion: The results obtained with hTAB004 suggest that the 111In/225Ac-DOTA-hTAB004 combination has significant potential as a theranostic strategy in TNBC and merits further development toward clinical translation.

Microbe-MUC1 Crosstalk in Cancer-Associated Infections.

Infection-associated cancers account for ∼20% of all malignancies. Understanding the molecular mechanisms underlying infection-associated malignancies may help in developing diagnostic biomarkers and preventative vaccines against malignancy. During infection, invading microbes interact with host mucins lining the glandular epithelial cells and trigger inflammation. MUC1 is a transmembrane mucin glycoprotein that is present on the surface of almost all epithelial cells, and is known to interact with invading microbes. This interaction can trigger pro- or anti-inflammatory responses depending on the microbe and the cell type. In this review we summarize the mechanisms of microbe and MUC1 interactions, and highlight how MUC1 plays contrasting roles in different cells. We also share perspectives on future research that may support clinical advances in infection-associated cancers.

APE1 senses DNA single-strand breaks for repair and signaling.

DNA single-strand breaks (SSBs) represent the most abundant type of DNA damage. Unrepaired SSBs impair DNA replication and transcription, leading to cancer and neurodegenerative disorders. Although PARP1 and XRCC1 are implicated in the SSB repair pathway, it remains unclear how SSB repair and SSB signaling pathways are coordinated and regulated. Using Xenopus egg extract and in vitro reconstitution systems, here we show that SSBs are first sensed by APE1 to initiate 3'-5' SSB end resection, followed by APE2 recruitment to continue SSB end resection. Notably, APE1's exonuclease activity is critical for SSB repair and SSB signaling pathways. An APE1 exonuclease-deficient mutant identified in somatic tissue from a cancer patient highlighted the significance of APE1 exonuclease activity in cancer etiology. In addition, APE1 interacts with APE2 and PCNA, although PCNA is dispensable for APE1's exonuclease activity. Taken together, we propose a two-step APE1/APE2-mediated mechanism for SSB end resection that couples DNA damage response with SSB repair in a eukaryotic system.

Indomethacin enhances anti-tumor efficacy of a MUC1 peptide vaccine against breast cancer in MUC1 transgenic mice.

In recent years, vaccines against tumor antigens have shown potential for combating invasive cancers, including primary tumors and metastatic lesions. This is particularly pertinent for breast cancer, which is the second-leading cause of cancer-related death in women. MUC1 is a glycoprotein that is normally expressed on glandular epithelium, but is overexpressed and under-glycosylated in most human cancers, including the majority of breast cancers. This under-glycosylation exposes the MUC1 protein core on the tumor-associated form of the protein. We have previously shown that a vaccine consisting of MUC1 core peptides stimulates a tumor-specific immune response. However, this immune response is dampened by the immunosuppressive microenvironment within breast tumors. Thus, in the present study, we investigated the effectiveness of MUC1 vaccination in combination with four different drugs that inhibit different components of the COX pathway: indomethacin (COX-1 and COX-2 inhibitor), celecoxib (COX-2 inhibitor), 1-methyl tryptophan (indoleamine 2,3 dioxygenase inhibitor), and AH6809 (prostaglandin E2 receptor antagonist). These treatment regimens were explored for the treatment of orthotopic MUC1-expressing breast tumors in mice transgenic for human MUC1. We found that the combination of vaccine and indomethacin resulted in a significant reduction in tumor burden. Indomethacin did not increase tumor-specific immune responses over vaccine alone, but rather appeared to reduce the proliferation and increase apoptosis of tumor cells, thus rendering them susceptible to immune cell killing.

Modulating MUC1 Function on T Cells.

Immunotherapy is a promising approach to treating cancer. Mucin1 (MUC1), an epithelial glycoprotein, is hypo-glycosylated and overexpressed on epithelial cancers. This renders it a promising target for potential immunotherapeutic approaches. However, MUC1 has also been identified on T cells, which might complicate its potential as a target for immunotherapies.

Role of Microbiome in Modulating Immune Responses in Cancer.

The complex interactions between genes and the environment play important roles in disease susceptibility and progression. One of the chronic diseases that is affected by this gene-environment interplay is cancer. However, our knowledge about these environmental factors remains limited. The microorganisms that inhabit our bodies have recently been acknowledged to play a crucial role as an environmental factor, to which we are constantly exposed. Studies have revealed significant differences in the relative abundance of certain microbes in cancer cases compared with controls. It has been reported that changes in the composition of normal gut microbiota can increase/decrease cancer susceptibility and progression by diverse mechanisms including, but not limited to, inflammation-a well-known hallmark of carcinogenesis. The microbiota can also affect the response to various treatments including immunotherapy. The microbiome-immune-cancer axis will continue to provide insight into the basic mechanisms of carcinogenesis. In this review, we provide a brief understanding of the mechanisms by which microbiota affects cancer development, progression, and treatment.

CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth.

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells.

Combining the Specific Anti-MUC1 Antibody TAB004 and Lip-MSA-IL-2 Limits Pancreatic Cancer Progression in Immune Competent Murine Models of Pancreatic Ductal Adenocarcinoma.

Immunotherapy regimens have shown success in subsets of cancer patients; however, their efficacy against pancreatic ductal adenocarcinoma (PDA) remain unclear. Previously, we demonstrated the potential of TAB004, a monoclonal antibody targeting the unique tumor-associated form of MUC1 (tMUC1) in the early detection of PDA. In this study, we evaluated the therapeutic benefit of combining the TAB004 antibody with Liposomal-MSA-IL-2 in immune competent and human MUC1 transgenic (MUC1.Tg) mouse models of PDA and investigated the associated immune responses. Treatment with TAB004 + Lip-MSA-IL-2 resulted in significantly improved survival and slower tumor growth compared to controls in MUC1.Tg mice bearing an orthotopic PDA.MUC1 tumor. Similarly, in the spontaneous model of PDA that expresses human MUC1, the combination treatment stalled the progression of pancreatic intraepithelial pre-neoplastic (PanIN) lesion to adenocarcinoma. Treatment with the combination elicited a robust systemic and tumor-specific immune response with (a) increased percentages of systemic and tumor infiltrated CD45+CD11b+ cells, (b) increased levels of myeloperoxidase (MPO), (c) increased antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP), (d) decreased percentage of immune regulatory cells (CD8+CD69+ cells), and (e) reduced circulating levels of immunosuppressive tMUC1. We report that treatment with a novel antibody against tMUC1 in combination with a unique formulation of IL-2 can improve survival and lead to stable disease in appropriate models of PDA by reducing tumor-induced immune regulation and promoting recruitment of CD45+CD11b+ cells, thereby enhancing ADCC/ADCP.

uMUC1-Targeting Magnetic Resonance Imaging of Therapeutic Response in an Orthotropic Mouse Model of Colon Cancer.

PURPOSE: Noninvasive assessment of chemotherapeutic response in colon cancer would tremendously aid in therapeutic intervention of cancer patients and improve outcomes. The aim of the study was to evaluate the feasibility of a noninvasive assessment of chemotherapeutic response by magnetic resonance imaging utilizing underglycosylated mucin 1 (uMUC1) tumor antigen as a biomarker of therapeutic response in a colon cancer mouse model. PROCEDURES: The study was performed by applying molecular imaging approach based on targeting uMUC1 with specific dual-modality imaging probe (MN-EPPT). The probe consisted of dextran-coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1-specific peptide (EPPT) and was used for magnetic resonance imaging (MRI) and fluorescence optical imaging. An orthotopic murine model of colon cancer expressing human uMUC1 peptide (MC38 MUC1) was created along with the control model devoid of the antigen (MC38 neo). Animals received chemotherapy with 5-fluorouracil (5-FU) followed by MN-EPPT-enhanced MR and optical imaging. RESULTS: In vivo imaging of animals with uMUC1 expressing tumors after 5-FU therapy showed that the average deltaT2 was reduced by 7.27 ms (p = 0.045) compared with animals in control groups indicating lower accumulation of MN-EPPT caused by uMUC1 downregulation. In vivo optical imaging, biodistribution, and fluorescence microscopy confirmed the MRI findings. Interestingly, we found that the group of animals that did not respond to chemotherapy ("progressive disease" per RECIST) showed higher accumulation of MN-EPPT compared to the group of responders ("stable disease") consistent with proliferating tumor cells and increased antigen availability. CONCLUSIONS: We believe that in application to over 50 % of human cancers expressing uMUC1, our results could provide insight into overall assessment of therapeutic response based on its expression as defined by non-invasive MN-EPPT-enhanced MRI.