ABSTRACT
Brain metastasis (BrM) is a major threat to the survival of melanoma, breast, and lung cancer patients. Circulating tumor cells (CTCs) cross the blood-brain barrier (BBB) and sustain in the brain microenvironment. Genetic mutations and epigenetic modifications have been found to be critical in controlling key aspects of cancer metastasis. Metastasizing cells confront inflammation and gradually adapt in the unique brain microenvironment. Currently, it is one of the major areas that has gained momentum. Researchers are interested in the factors that modulate neuroinflammation during BrM. We review here various epigenetic factors and mechanisms modulating neuroinflammation and how this helps CTCs to adapt and survive in the brain microenvironment. Since epigenetic changes could be modulated by targeting enzymes such as histone/DNA methyltransferase, deacetylases, acetyltransferases, and demethylases, we also summarize our current understanding of potential drugs targeting various aspects of epigenetic regulation in BrM.
Subject(s)
Brain Neoplasms , Lung Neoplasms , Humans , Epigenesis, Genetic , Neuroinflammatory Diseases , Brain Neoplasms/genetics , Inflammation/genetics , Tumor Microenvironment/geneticsABSTRACT
Pancreatic cancer (PC) is a highly lethal malignancy with a dismal five-year survival rate. This is due to its asymptomatic nature, lack of reliable biomarkers, poor resectability, early metastasis, and high recurrence rate. Limited efficacies of current treatment modalities treatment-associated toxicity underscore the need for the development of immunotherapy-based approaches. For non-resectable, locally advanced metastatic PC, immunotherapy-based approaches including vaccines, antibody-targeted, immune checkpoint inhibition, CAR-T-cells, and adoptive T-cell transfer could be valuable additions to existing treatment modalities. Thus far, the vaccine candidates in PC have demonstrated modest immunological responses in different treatment modalities. The identification of tumor-associated antigens (TAA) and their successful implication in PC treatment is still a challenge. MUC4, a high molecular weight glycoprotein that functionally contributes to PC pathogenesis, is an attractive TAA. It is not detected in the normal pancreas; however, it is overexpressed in mouse and human pancreatic tumors. The recombinant MUC4 domain, as well as predicted immunogenic T-cell epitopes, elicited cellular and humoral anti-MUC4 response, suggesting its ulility as a vaccine candidate for PC therapy. Existence of PC-associated MUC4 splice variants, autoantibodies against overexpressed and aberrantly glycosylated MUC4 and presence of T-cell clones against the mutations present in MUC4 further reinforce its significance as a tumor antigen for vaccine development. Herein, we review the significance of MUC4 as a tumor antigen in PC immunotherapy and discuss both, the development and challenges associated with MUC4 based immunotherapy. Lastly, we will present our perspective on MUC4 antigenicity for the future development of MUC4-based PC immunotherapy.
Subject(s)
Antigens, Neoplasm/immunology , Immunotherapy , Mucin-4/immunology , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/therapy , Animals , Antigens, Neoplasm/genetics , Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Computational Biology/methods , Epitopes , Humans , Immunotherapy/adverse effects , Immunotherapy/methods , Mucin-4/antagonists & inhibitors , Mucin-4/genetics , Mutation , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by high resistance and poor response to chemotherapy. In addition, the poorly immunogenic pancreatic tumors constitute an immunosuppressive tumor microenvironment (TME) that render immunotherapy-based approaches ineffective. Understanding the mechanisms of therapy resistance, identifying new targets, and developing effective strategies to overcome resistance can significantly impact the management of PDAC patients. Chemokines are small soluble factors that are significantly deregulated during PDAC pathogenesis, contributing to tumor growth, metastasis, immune cell trafficking, and therapy resistance. Thus far, different chemokine pathways have been explored as therapeutic targets in PDAC, with some promising results in recent clinical trials. Particularly, immunotherapies such as immune check point blockade therapies and CAR-T cell therapies have shown promising results when combined with chemokine targeted therapies. Considering the emerging pathological and clinical significance of chemokines in PDAC, we reviewed major chemokine-regulated pathways leading to therapy resistance and the ongoing endeavors to target chemokine signaling in PDAC. This review discusses the role of chemokines in regulating therapy resistance in PDAC and highlights the continuing efforts to target chemokine-regulated pathways to improve the efficacy of various treatment modalities.
Subject(s)
Carcinoma, Pancreatic Ductal , Chemokines , Drug Resistance, Neoplasm , Pancreatic Neoplasms , Humans , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Chemokines/genetics , Chemokines/immunology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/immunology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/immunology , Pancreatic NeoplasmsABSTRACT
Immunosuppression is a hallmark of pancreatic ductal adenocarcinoma (PDAC), contributing to early metastasis and poor patient survival. Compared to the localized tumors, current standard-of-care therapies have failed to improve the survival of patients with metastatic PDAC, that necessecitates exploration of novel therapeutic approaches. While immunotherapies such as immune checkpoint blockade (ICB) and therapeutic vaccines have emerged as promising treatment modalities in certain cancers, limited responses have been achieved in PDAC. Therefore, specific mechanisms regulating the poor response to immunotherapy must be explored. The immunosuppressive microenvironment driven by oncogenic mutations, tumor secretome, non-coding RNAs, and tumor microbiome persists throughout PDAC progression, allowing neoplastic cells to grow locally and metastasize distantly. The metastatic cells escaping the host immune surveillance are unique in molecular, immunological, and metabolic characteristics. Following chemokine and exosomal guidance, these cells metastasize to the organ-specific pre-metastatic niches (PMNs) constituted by local resident cells, stromal fibroblasts, and suppressive immune cells, such as the metastasis-associated macrophages, neutrophils, and myeloid-derived suppressor cells. The metastatic immune microenvironment differs from primary tumors in stromal and immune cell composition, functionality, and metabolism. Thus far, multiple molecular and metabolic pathways, distinct from primary tumors, have been identified that dampen immune effector functions, confounding the immunotherapy response in metastatic PDAC. This review describes major immunoregulatory pathways that contribute to the metastatic progression and limit immunotherapy outcomes in PDAC. Overall, we highlight the therapeutic vulnerabilities attributable to immunosuppressive factors and discuss whether targeting these molecular and immunological "hot spots" could improve the outcomes of PDAC immunotherapies.
Subject(s)
Adenocarcinoma , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Immunosuppression Therapy , Immunotherapy , Tumor MicroenvironmentABSTRACT
BACKGROUND & AIMS: Tumor-microenvironment factors and cancer stem cells (CSCs) play a critical role in the aggressiveness of pancreatic cancer (PC). However, the degree to which tumor-microenvironment factors promote stemness remains unexplored. Here, we examined whether cancer-associated fibroblasts (CAFs) promote CSC features in PC. METHODS: PC cells were treated long-term (30, 60, and 90 days) with conditioned media (CM)-derived from normal human fibroblasts (NFs) and CAFs. The stemness features of tumorsphere formation and stemness populations, along with CSCs markers, were analyzed using 2-dimensional and 3-dimensional sodium alginate bead-based co-culture models. Immunohistochemistry and immunofluorescence staining were performed for CSCs and fibroblast markers in autochthonous KrasG12D/+; Trp53R172H/+; Pdx1-Cre mice and human pancreatic tumors. Polymerase chain reaction array and gene knockdown were performed to identify the mechanism of stemness enrichment. RESULTS: Long-term treatment of PC cells with CAF-CM enriched stemness, as indicated by significantly higher CD44+, ALDH+, and AF+ populations in PC cells. Increased tumorsphere formation and elevated CSC, self-renewal, and drug-resistance markers in CAF-CM-treated PC cells were observed. In addition, CAFs co-cultured with PC cells in the 3-dimensional model showed a substantial increase in stemness features. CD44 and α-smooth muscle actin were positively correlated and their expressions progressively increased from the early to late stages of KrasG12D/+; Trp53R172H/+; Pdx1-Cre mouse and human pancreatic tumors. Osteopontin/secreted phosphoprotein 1 was identified as the top differentially overexpressed gene in CAF-CM-treated PC cells and knockdown of osteopontin/secreted phosphoprotein 1 significantly reduced stemness characteristics in CAF-CM-treated PC cells. CONCLUSIONS: Our data uncovered novel insight into the interplay between CAF and enrichment of stemness population through the osteopontin/secreted phosphoprotein 1-CD44 axis in PC.
Subject(s)
Cancer-Associated Fibroblasts/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Hyaluronan Receptors/metabolism , Neoplastic Stem Cells/metabolism , Osteopontin/metabolism , Pancreatic Neoplasms/metabolism , Tumor Microenvironment , Animals , Cancer-Associated Fibroblasts/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Coculture Techniques , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Humans , Hyaluronan Receptors/genetics , Male , Mice, Nude , Mice, Transgenic , Neoplasm Invasiveness , Neoplastic Stem Cells/pathology , Osteopontin/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Paracrine Communication , Phenotype , Signal TransductionABSTRACT
Mucins (MUC) protect epithelial barriers from environmental insult to maintain homeostasis. However, their aberrant overexpression and glycosylation in various malignancies facilitate oncogenic events from inception to metastasis. Mucin-associated sialyl-Tn (sTn) antigens bind to various receptors present on the dendritic cells (DCs), macrophages, and natural killer (NK) cells, resulting in overall immunosuppression by either receptor masking or inhibition of cytolytic activity. MUC1-mediated interaction of tumor cells with innate immune cells hampers cross-presentation of processed antigens on MHC class I molecules. MUC1 and MUC16 bind siglecs and mask Toll-like receptors (TLRs), respectively, on DCs promoting an immature DC phenotype that in turn reduces T cell effector functions. Mucins, such as MUC1, MUC2, MUC4, and MUC16, interact with or form aggregates with neutrophils, macrophages, and platelets, conferring protection to cancer cells during hematological dissemination and facilitate their spread and colonization to the metastatic sites. On the contrary, poor glycosylation of MUC1 and MUC4 at the tandem repeat region (TR) generates cancer-specific immunodominant epitopes. The presence of MUC16 neo-antigen-specific T cell clones and anti-MUC1 antibodies in cancer patients suggests that mucins can serve as potential targets for developing cancer therapeutics. The present review summarizes the molecular events involved in mucin-mediated immunomodulation, and metastasis, as well as the utility of mucins as targets for cancer immunotherapy and radioimmunotherapy.
Subject(s)
Mucins/immunology , Neoplasms/immunology , Neoplasms/pathology , Animals , Humans , Immunomodulation , Neoplasm MetastasisABSTRACT
The SYBR green I (SG) dye-based fluorescence assay for screening antimalarial compounds is based on direct quantitation of parasite DNA. We show that DNA-interacting cationic cell-penetrating peptides (CPPs) and intercalating agents compete with SG dye to bind to DNA. Therefore, readouts of this assay, unlike those of the [(3)H]hypoxanthine incorporation assay, for the antimalarial activity of the above DNA binding agents may be erroneous. In the case of CPPs, false readouts can be improved by the removal of excess peptides.
Subject(s)
Antimalarials/chemistry , Biological Assay/methods , Cell-Penetrating Peptides/chemistry , Intercalating Agents/chemistry , Organic Chemicals/chemistry , Benzothiazoles , DNA/chemistry , Diamines , Fluorescence , QuinolinesABSTRACT
Early detection significantly correlates with improved survival in cancer patients. So far, a limited number of biomarkers have been validated to diagnose cancers at an early stage. Considering the leading cancer types that contribute to more than 50% of deaths in the USA, we discuss the ongoing endeavors toward early detection of lung, breast, ovarian, colon, prostate, liver, and pancreatic cancers to highlight the significance of mucin glycoproteins in cancer diagnosis. As mucin deregulation is one of the earliest events in most epithelial malignancies following oncogenic transformation, these high-molecular-weight glycoproteins are considered potential candidates for biomarker development. The diagnostic potential of mucins is mainly attributed to their deregulated expression, altered glycosylation, splicing, and ability to induce autoantibodies. Secretory and shed mucins are commonly detected in patients' sera, body fluids, and tumor biopsies. For instance, CA125, also called MUC16, is one of the biomarkers implemented for the diagnosis of ovarian cancer and is currently being investigated for other malignancies. Similarly, MUC5AC, a secretory mucin, is a potential biomarker for pancreatic cancer. Moreover, anti-mucin autoantibodies and mucin-packaged exosomes have opened new avenues of biomarker development for early cancer diagnosis. In this review, we discuss the diagnostic potential of mucins in epithelial cancers and provide evidence and a rationale for developing a mucin-based biomarker panel for early cancer detection.
ABSTRACT
Aberrantly expressed onco-mucin 16 (MUC16) and its post-cleavage generated surface tethered carboxy-terminal (MUC16-Cter) domain are strongly associated with poor prognosis and lethality of pancreatic (PC) and non-small cell lung cancer (NSCLC). To date, most anti-MUC16 antibodies are directed towards the extracellular domain of MUC16 (CA125), which is usually cleaved and shed in the circulation hence obscuring antibody accessibility to the cancer cells. Herein, we establish the utility of targeting a post-cleavage generated, surface-tethered oncogenic MUC16 carboxy-terminal (MUC16-Cter) domain by using a novel chimeric antibody in human IgG1 format, ch5E6, whose epitope expression directly correlates with disease severity in both cancers. ch5E6 binds and interferes with MUC16-associated oncogenesis, suppresses the downstream signaling pFAK(Y397)/p-p70S6K(T389)/N-cadherin axis and exert antiproliferative effects in cancer cells, 3D organoids, and tumor xenografts of both PC and NSCLC. The robust clinical correlations observed between MUC16 and N-cadherin in patient tumors and metastatic samples imply ch5E6 potential in targeting a complex and significantly occurring phenomenon of epithelial to mesenchymal transition (EMT) associated with disease aggressiveness. Our study supports evaluating ch5E6 with standard-of-care drugs, to potentially augment treatment outcomes in malignancies inflicted with MUC16-associated poor prognosis.
ABSTRACT
Delivery of therapeutic agents in pancreatic cancer (PC) is impaired due to its hypovascular and desmoplastic tumor microenvironment. The Endothelin (ET)-axis is the major regulator of vasomotor tone under physiological conditions and is highly upregulated in multiple cancers. We investigated the effect of dual endothelin receptor antagonist bosentan on perfusion and macromolecular transport in a PC cell-fibroblast co-implantation tumor model using Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI). Following bosentan treatment, the contrast enhancement ratio and wash-in rates in tumors were two- and nine times higher, respectively, compared to the controls, whereas the time to peak was significantly shorter (7.29 ± 1.29 min v/s 22.08 ± 5.88 min; p = 0.04). Importantly, these effects were tumor selective as the magnitudes of change for these parameters were much lower in muscles. Bosentan treatment also reduced desmoplasia and improved intratumoral distribution of high molecular weight FITC-dextran. Overall, these findings support that targeting the ET-axis can serve as a potential strategy to selectively enhance tumor perfusion and improve the delivery of therapeutic agents in pancreatic tumors.
Subject(s)
Endothelin Receptor Antagonists , Pancreatic Neoplasms , Bosentan , Endothelin Receptor Antagonists/pharmacology , Endothelin Receptor Antagonists/therapeutic use , Endothelins , Humans , Pancreatic Neoplasms/drug therapy , Perfusion , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Tumor Microenvironment , Pancreatic NeoplasmsABSTRACT
Pancreatic tumors are highly desmoplastic and immunosuppressive. Delivery and distribution of drugs within pancreatic tumors are compromised due to intrinsic physical and biochemical stresses that lead to increased interstitial fluid pressure, vascular compression, and hypoxia. Immunotherapy-based approaches, including therapeutic vaccines, immune checkpoint inhibition, CAR-T cell therapy, and adoptive T cell therapies, are challenged by an immunosuppressive tumor microenvironment. Together, extensive fibrosis and immunosuppression present major challenges to developing treatments for pancreatic cancer. In this context, nanoparticles have been extensively studied as delivery platforms and adjuvants for cancer and other disease therapies. Recent advances in nanotechnology have led to the development of multiple nanocarrier-based formulations that not only improve drug delivery but also enhance immunotherapy-based approaches for pancreatic cancer. This review discusses and critically analyzes the novel nanoscale strategies that have been used for drug delivery and immunomodulation to improve treatment efficacy, including newly emerging immunotherapy-based approaches. This review also presents important perspectives on future research directions that will guide the rational design of novel and robust nanoscale platforms to treat pancreatic tumors, particularly with respect to targeted therapies and immunotherapies. These insights will inform the next generation of clinical treatments to help patients manage this debilitating disease and enhance survival rates.
Subject(s)
Pancreatic Neoplasms , Humans , Immunologic Factors , Immunotherapy , Immunotherapy, Adoptive , Pancreatic Neoplasms/therapy , Tumor Microenvironment , Pancreatic NeoplasmsABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, as it commonly metastasizes to the liver resulting in an overall poor prognosis. However, the molecular mechanism involved in liver metastasis remains poorly understood. Here, we aimed to identify the MUC16-mediated molecular mechanism of PDAC-liver metastasis. Previous studies demonstrated that MUC16 and its C-terminal (Cter) domain are involved in the aggressiveness of PDAC. In this study, we observed MUC16 and its Cter expression significantly high in human PDAC tissues, PDAC organoids, and metastatic liver tissues, while no expression was observed in normal pancreatic tissues using IHC and immunofluorescence (IFC) analyses. MUC16 knockdown in SW1990 and CD18/HPAF PDAC cells significantly decreased the colony formation, migration, and endothelial/p-selectin binding. In contrast, MUC16-Cter ectopic overexpression showed significantly increased colony formation and motility in MiaPaCa2 pancreatic cancer cells. Interestingly, MUC16 promoted cell survival and colonization in the liver, mimicking an ex vivo environment. Furthermore, MUC16 enhanced liver metastasis in the in vivo mouse model. Our integrated analyses of RNA-sequencing suggested that MUC16 alters Neuropilin-2 (NRP2) and cell adhesion molecules in pancreatic cancer cells. Furthermore, we identified that MUC16 regulated NRP2 via JAK2/STAT1 signaling in PDAC. NRP2 knockdown in MUC16-overexpressed PDAC cells showed significantly decreased cell adhesion and migration. Overall, the findings indicate that MUC16 regulates NRP2 and induces metastasis in PDAC. IMPLICATIONS: This study shows that MUC16 plays a critical role in PDAC liver metastasis by mediating NRP2 regulation by JAK2/STAT1 axis, thereby paving the way for future therapy efforts for metastatic PDAC.
Subject(s)
Adenocarcinoma , Carcinoma, Pancreatic Ductal , Liver Neoplasms , Neuropilin-2 , Pancreatic Neoplasms , Adenocarcinoma/pathology , Animals , CA-125 Antigen/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Membrane Proteins/metabolism , Mice , Neoplasm Metastasis , Neuropilin-2/genetics , Pancreatic Neoplasms/pathology , Pancreatic NeoplasmsABSTRACT
BACKGROUND: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells. METHODS: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient's clinical study. FINDINGS: FDPS overexpression in PDAC tissues and cells (P < 0.01 and P < 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P < 0.05, P < 0.01, and P < 0.001) and in vivo (P < 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment. INTERPRETATION: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers. FUNDING: National Institutes of Health (P50, P01, and R01).
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Animals , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/radiotherapy , Cell Line, Tumor , Cell Proliferation , DNA Damage , Gene Expression Regulation, Neoplastic , Geranyltranstransferase/genetics , Geranyltranstransferase/metabolism , Humans , Mice , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/radiotherapy , Signal Transduction , Tumor Microenvironment/genetics , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
MUC16, membrane-bound mucin, plays an oncogenic role in pancreatic ductal adenocarcinoma (PDAC). However, the pathological role of MUC16 in the PDAC progression, tumor microenvironment, and metastasis in cooperation with KrasG12D and Trp53R172H mutations remains unknown. Deletion of Muc16 with activating mutations KrasG12D/+ and Trp53R172H/+ in mice significantly decreased progression and prolonged overall survival in KrasG12D/+; Trp53R172H/+; Pdx-1-Cre; Muc16-/- (KPCM) and KrasG12D/+; Pdx-1-Cre; Muc16-/- (KCM), as compared to KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) and KrasG12D/+; Pdx-1-Cre (KC) mice, respectively. Muc16 knockout pancreatic tumor (KPCM) displays decreased tumor microenvironment factors and significantly reduced incidence of liver and lung metastasis compared to KPC. Furthermore, in silico data analysis showed a positive correlation of MUC16 with activated stroma and metastasis-associated genes. KPCM mouse syngeneic cells had significantly lower metastatic and endothelial cell binding abilities than KPC cells. Similarly, KPCM organoids significantly decreased the growth rate compared to KPC organoids. Interestingly, RNA-seq data revealed that the cytoskeletal proteins Actg2, Myh11, and Pdlim3 were downregulated in KPCM tumors. Further knockdown of these genes showed reduced metastatic potential. Overall, our results demonstrate that Muc16 alters the tumor microenvironment factors during pancreatic cancer progression and metastasis by changing the expression of Actg2, Myh11, and Pdlim3 genes.
Subject(s)
Carcinoma, Pancreatic Ductal , Mucins , Pancreatic Neoplasms , Animals , Mice , Carcinogenesis , Carcinoma, Pancreatic Ductal/pathology , Mucins/metabolism , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Tumor Microenvironment/genetics , Pancreatic NeoplasmsABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy that is characterized by early metastasis, low resectability, high recurrence, and therapy resistance. The experimental mouse models have played a central role in understanding the pathobiology of PDAC and in the preclinical evaluation of various therapeutic modalities. Different mouse models with targetable pathological hallmarks have been developed and employed to address the unique challenges associated with PDAC progression, metastasis, and stromal heterogeneity. Over the years, mouse models have evolved from simple cell line-based heterotopic and orthotopic xenografts in immunocompromised mice to more complex and realistic genetically engineered mouse models (GEMMs) involving multi-gene manipulations. The GEMMs, mostly driven by KRAS mutation(s), have been widely accepted for therapeutic optimization due to their high penetrance and ability to recapitulate the histological, molecular, and pathological hallmarks of human PDAC, including comparable precursor lesions, extensive metastasis, desmoplasia, perineural invasion, and immunosuppressive tumor microenvironment. Advanced GEMMs modified to express fluorescent proteins have allowed cell lineage tracing to provide novel insights and a new understanding about the origin and contribution of various cell types in PDAC pathobiology. The syngeneic mouse models, GEMMs, and target-specific transgenic mice have been extensively used to evaluate immunotherapies and study therapy-induced immune modulation in PDAC yielding meaningful results to guide various clinical trials. The emerging mouse models for parabiosis, hepatic metastasis, cachexia, and image-guided implantation, are increasingly appreciated for their high translational significance. In this article, we describe the contribution of various experimental mouse models to the current understanding of PDAC pathobiology and their utility in evaluating and optimizing therapeutic modalities for this lethal malignancy.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/therapy , Immunotherapy , Neoplasms, Experimental/therapy , Pancreatic Neoplasms/therapy , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm , Genetic Predisposition to Disease , Mice, Transgenic , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phenotype , Species Specificity , Tumor Microenvironment , Xenograft Model Antitumor AssaysABSTRACT
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 ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß 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 affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß 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.
Subject(s)
Mucin-4/genetics , Mucin-4/metabolism , Protein Subunits , Recombinant Proteins , Cloning, Molecular , Gene Expression , Gene Order , Humans , Mass Spectrometry , Models, Molecular , Mucin-4/chemistry , Mucin-4/isolation & purification , Plasmids/genetics , Protein Binding , Structure-Activity RelationshipABSTRACT
Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4ß-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4ß protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4ß-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4ß-loaded nanoparticles generated MUC4ß-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4ß vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.
Subject(s)
Antigens, Neoplasm/chemistry , Cancer Vaccines/chemistry , Mucin-4/chemistry , Nanoparticles , Pancreatic Neoplasms/chemistry , Polyanhydrides/chemistry , Animals , Drug Carriers , Epitopes , Humans , MiceABSTRACT
Mucus serves as the chief protective barrier against pathogenic and mechanical insults in respiratory, gastrointestinal, and urogenital tracts. Altered mucin expression, the major component of mucus, in conjunction with differential glycosylation has been strongly associated with both benign and malignant pathologies of colon. Mucins and their associated glycans arbitrate their impact sterically as well as mechanically by altering molecular and microbial spectrum during pathogenesis. Mucin expression in normal and pathological conditions is regulated by nonspecific (dietary factors and gut microbiota) and specific (epigenetic and transcriptional) modulators. Further, recent studies highlight the impact of altering mucin glycome (cancer-associated carbohydrate antigens including Tn, Sialyl-Tn, Sialyl-Lew A, and Sialyl-Lewis X) on host immunomodulation, antitumor immunity, as well as gut microbiota. In light of emerging literature, the present review article digs into the impact of structural organization and of expressional and glycosylation alteration of mucin family members on benign and malignant pathologies of colorectal cancer.
ABSTRACT
Endothelin-1 (ET-1) and its two receptors, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) exhibit deregulated overexprerssion in pancreatic ductal adenocarcinoma (PDAC) and pancreatitis. We examined the expression pattern of endothelin (ET) axis components in the murine models of chronic and acute inflammation in the presence or absence of oncogenic K-ras. While the expression of endothelin converting enzyme-1 (ECE-1), ET-1, ETAR and ETBR in the normal pancreas is restricted predominantly to the islet cells, progressive increase of ET receptors in ductal cells and stromal compartment is observed in the KC model (Pdx-1 Cre; K-rasG12D) of PDAC. In the murine pancreas harboring K-rasG12D mutation (KC mice), following acute inflammation induced by cerulein, increased ETAR and ETBR expression is observed in the amylase and CK19 double positive cells that represent cells undergoing pancreatic acinar to ductal metaplasia (ADM). As compared to the wild type (WT) mice, cerulein treatment in KC mice resulted in significantly higher levels of ECE-1, ET-1, ETAR and ETBR, transcripts in the pancreas. Similarly, in response to cigarette smoke-induced chronic inflammation, the expression of ET axis components is significantly upregulated in the pancreas of KC mice as compared to the WT mice. In addition to the expression in the precursor pancreatic intraepithelial neoplasm (PanIN lesions) in cigarette smoke-exposure model and metaplastic ducts in cerulein-treatment model, ETAR and ETBR expression is also observed in infiltrating F4/80 positive macrophages and α-SMA positive fibroblasts and high co-localization was seen in the presence of oncogenic K-ras. In conclusion, both chronic and acute pancreatic inflammation in the presence of oncogenic K-ras contribute to sustained upregulation of ET axis components in the ductal and stromal cells suggesting a potential role of ET axis in the initiation and progression of PDAC.
Subject(s)
Endothelin-1/genetics , Inflammation/genetics , Pancreatic Neoplasms/genetics , Pancreatitis/genetics , Receptor, Endothelin A/genetics , Receptor, Endothelin B/genetics , Amylases/genetics , Animals , Ceruletide/toxicity , Disease Models, Animal , Endothelin-Converting Enzymes/genetics , Gene Expression Regulation/genetics , Humans , Inflammation/chemically induced , Inflammation/pathology , Mice , Oncogenes/genetics , Pancreatic Neoplasms/pathology , Pancreatitis/chemically induced , Pancreatitis/pathology , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
Brain metastasis (BrM) remains a significant cause of cancer-related mortality in epidermal growth factor receptor 2-positive (ERBB2+) breast cancer (BC) patients. We proposed here that a combination treatment of irreversible tyrosine kinase inhibitor neratinib (NER) and the c-MET inhibitor cabozantinib (CBZ) could prevent brain metastasis. To address this, we first tested the combination treatment of NER and CBZ in the brain-seeking ERBB2+ cell lines SKBrM3 and JIMT-1-BR3, and in ERBB2+ organoids that expressed the c-MET/ERBB1 axis. Next, we developed and characterized an orthotopic mouse model of spontaneous BrM and evaluated the therapeutic effect of CBZ and NER in vivo. The combination treatment of NER and CBZ significantly inhibited proliferation and migration in ERBB2+ cell lines and reduced the organoid growth in vitro. Mechanistically, the combination treatment of NER and CBZ substantially inhibited ERK activation downstream of the c-MET/ERBB1 axis. Orthotopically implanted SKBrM3+ cells formed primary tumor in the mammary fat pad and spontaneously metastasized to the brain and other distant organs. Combination treatment with NER and CBZ inhibited primary tumor growth and predominantly prevented BrM. In conclusion, the orthotopic model of spontaneous BrM is clinically relevant, and the combination therapy of NER and CBZ might be a useful approach to prevent BrM in BC.