The Th2 cytokines, interleukin (IL)-4 and -13, are structurally and functionally related. They regulate immune responses and the immune microenvironment, not only under normal physiological ...conditions, but also in cancer. Both cytokines bind to their high-affinity receptors and form various configurations of receptor subtypes. We and others have reported that IL-4 and IL-13 bind to IL-4Rα and IL-13Rα1 chains, forming functional receptors in cancer cells. IL-13 also binds with high affinity to a private chain IL-13Rα2. After forming ligand-receptor complexes, both cytokines initiate signal transduction and mediate biological effects, such as tumor proliferation, cell survival, cell adhesion and metastasis. In certain cancers, the presence of these cytokine receptors may serve as biomarkers of cancer aggressiveness.
In a series of studies, we reported that overexpression of IL-4 and IL-13 receptors on cancer cells provides targets for therapeutic agents for cancer therapy. In addition, both of these cytokines and their receptors have been shown to play important roles in modulating the immune system for tumor growth. IL-4, IL-13 and their receptors seem to play a role in cancer stem cells and provide unique targets to eradicate these cells. In this review article, we summarize some of the important attributes of IL-4 and IL-13 receptors in cancer biology and discuss pre-clinical and clinical studies pertaining to recombinant immunotoxins designed to target these receptors.
Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, ...we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.
Previously, we have demonstrated that a variety of human cancers including the ovarian cancer express IL‐13Rα2, a high affinity receptor for IL‐13. Herein, we have examined if IL‐13 regulates ...invasion and metastasis of ovarian cancer through IL‐13Rα2 in vitro and in vivo in animal models of human ovarian cancer. We tested cell invasion and protease activity in IL‐13Rα2‐overexpressing and IL‐13Rα2‐negative ovarian tumor cell lines. IL‐13 treatment significantly augmented both cell invasion and enzyme activities in only IL‐13Rα2‐positive cells but not in IL‐13Rα2‐negative cells in vitro. Mechanistically, IL‐13 enhanced ERK1/2, AP‐1 and MMP activities only in IL‐13Rα2‐positive cells but not in IL‐13Rα2‐negative cells. In contrast, other signaling pathways such as IRS1/2, PI3K and AKT do not seem to be involved in IL‐13 induced signaling in ovarian cancer cell lines. Highly specific inhibitors for MMP and AP‐1 efficiently inhibited both invasion and protease activities without impacting the basal level invasion and protease activities in vitro. In orthotopic animal model of human ovarian cancer, IL‐13Rα2‐positive tumors metastasized to lymph nodes and peritoneum earlier than IL‐13Rα2‐negative tumors. Interestingly, the IL‐13Rα2‐positive tumor bearing mice died earlier than mice with IL‐13Rα2‐negative tumor. Intraperitoneal injection of IL‐13 further shortened survival of IL‐13Rα2‐positive tumor bearing mice compared to IL‐13Rα2‐negative tumor mice. IL‐13Rα2‐positive tumors and lymph node metastasis expressed higher levels of MMPs and higher ERK1/2 activation compared to IL‐13Rα2‐negative tumors. Taken together, IL‐13Rα2 is involved in cancer metastasis through activation of ERK/AP‐1 and that targeting IL‐13Rα2 might not only directly kill primary tumors but also prevent cancer metastasis.
Previously, we have demonstrated that Interleukin 13 receptor alpha 2 (IL-13Rα2) is overexpressed in approximate 78% Glioblastoma multiforme (GBM) samples. We have also demonstrated that IL-13Rα2 can ...serve as a target for cancer immunotherapy in several pre-clinical and clinical studies. However, the significance of overexpression of IL-13Rα2 in GBM and astrocytoma and signaling through these receptors is not known. IL-13 can signal through IL-13R via JAK/STAT and AP-1 pathways in certain cell lines including some tumor cell lines. Herein, we have investigated a role of IL-13/IL-13Rα2 axis in signaling through AP-1 transcription factors in human glioma samples in situ.
We examined the activation of AP-1 family of transcription factors (c-Jun, Fra-1, Jun-D, c-Fos, and Jun-B) after treating U251, A172 (IL-13Rα2 +ve) and T98G (IL-13Rα2 -ve) glioma cell lines with IL-13 by RT-qPCR, and immunocytochemistry (ICC). We also performed colorimetric ELISA based assay to determine AP-1 transcription factor activation in glioma cell lines. Furthermore, we examined the expression of AP-1 transcription factors in situ in GBM and astrocytoma specimens by multiplex-immunohistochemistry (IHC). Student t test and ANOVA were used for statistical analysis of the results.
We have demonstrated up-regulation of two AP-1 transcription factors (c-Jun and Fra-1) at mRNA and protein levels upon treatment with IL-13 in IL-13Rα2 positive but not in IL-13Rα2 negative glioma cell lines. Both transcription factors were also overexpressed in patient derived GBM specimens, however, in contrast to GBM cell lines, c-Fos is also overexpressed in patient derived specimens. Astrocytoma specimens showed lesser extent of immunostaining for IL-13Rα2 and three AP-1 factors compared to GBM specimens. By transcription factor activation assay, we demonstrated that AP-1 transcription factors (C-Jun and Fra-1) were activated upon treatment of IL-13Rα2 + GBM cell lines but not IL-13Rα2 - GBM cell line with IL-13. Our results demonstrate functional activity of AP-1 transcription factor in GBM cell lines in response to IL-13.
These results indicate that IL-13/IL-13Rα2 axis can mediate signal transduction in situ via AP-1 pathway in GBM and astrocytoma and may serve as a new target for GBM immunotherapy.
Abstract
Background
Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as ...cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products.
Methods
To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with
89
Zirconium-oxine (
89
Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The
89
Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells.
Results
We observed that radiolabeling of CAR-T cells with
89
Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells.
Conclusions
Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with
89
Zr-oxine retain critical product attributes and suggest
89
Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.
Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three ...scaffold domains, hemispherical S2 scaffolds and smaller S1B and S1A scaffolds. The caveolin scaffolding domain (CSD) is a highly conserved hydrophobic region that mediates interaction of CAV1 with multiple effector molecules. F92A/V94A mutation disrupts CSD function, however the structural impact of CSD mutation on caveolae or scaffolds remains unknown. Here, SMLM network analysis quantitatively shows that expression of the CAV1 CSD F92A/V94A mutant in CRISPR/Cas CAV1 knockout MDA-MB-231 breast cancer cells reduces the size and volume and enhances the elongation of caveolae and scaffold domains, with more pronounced effects on S2 and S1B scaffolds. Convex hull analysis of the outer surface of the CAV1 point clouds confirms the size reduction of CSD mutant CAV1 blobs and shows that CSD mutation reduces volume variation amongst S2 and S1B CAV1 blobs at increasing shrink values, that may reflect retraction of the CAV1 N-terminus towards the membrane, potentially preventing accessibility of the CSD. Detection of point mutation-induced changes to CAV1 domains highlights the utility of SMLM network analysis for mesoscale structural analysis of oligomers in their native environment.
Macromolecular complexes exhibit reduced diffusion in biological membranes; however, the physiological consequences of this characteristic of plasma membrane domain organization remain elusive. We ...report that competition between the galectin lattice and oligomerized caveolin-1 microdomains for epidermal growth factor (EGF) receptor (EGFR) recruitment regulates EGFR signaling in tumor cells. In mammary tumor cells deficient for Golgi β1,6N-acetylglucosaminyltransferase V (Mgat5), a reduction in EGFR binding to the galectin lattice allows an increased association with stable caveolin-1 cell surface microdomains that suppresses EGFR signaling. Depletion of caveolin-1 enhances EGFR diffusion, responsiveness to EGF, and relieves Mgat5 deficiency-imposed restrictions on tumor cell growth. In Mgat5⁺/⁺ tumor cells, EGFR association with the galectin lattice reduces first-order EGFR diffusion rates and promotes receptor interaction with the actin cytoskeleton. Importantly, EGFR association with the lattice opposes sequestration by caveolin-1, overriding its negative regulation of EGFR diffusion and signaling. Therefore, caveolin-1 is a conditional tumor suppressor whose loss is advantageous when β1,6GlcNAc-branched N-glycans are below a threshold for optimal galectin lattice formation.
Expression of Caveolin-1 (Cav1), a key component of cell surface caveolae, is elevated in prostate cancer (PCa) and associated with PCa metastasis and a poor prognosis for PCa patients. Polymerase I ...and Transcript Release Factor (PTRF)/cavin-1 is a cytoplasmic protein required for Cav1-dependent formation of caveolae. Expression of PTRF reduces the motility of PC3 cells, a metastatic prostate cancer cell line that endogenously expresses abundant Cav1 but no PTRF and no caveolae, suggesting a role for non-caveolar Cav1 domains, or Cav1 scaffolds, in PCa cell migration. Tyrosine phosphorylated Cav1 (pCav1) functions in concert with Galectin-3 (Gal3) and the galectin lattice to stabilize focal adhesion kinase (FAK) within focal adhesions (FAs) and promote cancer cell motility. However, whether PTRF regulation of Cav1 function in PCa cell migration is related to Gal3 expression and functionality has yet to be determined. Here we show that PTRF expression in PC3 cells reduces FAK stabilization in focal adhesions and reduces cell motility without affecting pCav1 levels. Exogenous Gal3 stabilized FAK in focal adhesions of PTRF-expressing cells and restored cell motility of PTRF-expressing PC3 cells to levels of PC3 cells in a dose-dependent manner, with an optimal concentration of 2 µg/ml. Exogenous Gal3 stabilized FAK in focal adhesions of Gal3 knockdown PC3 cells but not in Cav1 knockdown PC3 cells. Cav1 knockdown also prevented Gal3 rescue of FA-associated FAK stabilization in PTRF-expressing PC3 cells. Our data support a role for PTRF/cavin-1, through caveolae formation, as an attenuator of the non-caveolar functionality of Cav1 in Gal3-Cav1 signalling and regulation of focal adhesion dynamics and cancer cell migration.
Glycoprotein 78 (Gp78) is a critical E3 ubiquitin ligase in endoplasmic reticulum-associated degradation. Overexpression of Flag-tagged Gp78 (Flag-gp78), but not Flag-gp78 mutated in its RING-finger ...domain (Flag-RINGmut) with deficient ubiquitin ligase activity, induces mitochondrial fragmentation and ubiquitination and proteasome-dependent degradation of the mitofusin (Mfn) mitochondrial fusion factors Mfn1/Mfn2. After mitochondrial depolarization with carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flag-gp78 induced a threefold loss of depolarized mitochondria and significant loss of the inner mitochondrial protein OxPhosV. Flag-gp78-dependent loss of OxPhosV, but not Mfn1 or Mfn2, was prevented by small interfering RNA (siRNA) knockdown of the autophagy protein Atg5 in CCCP-treated cells. Gp78-induced mitophagy required ubiquitin ligase activity, as it is not observed upon transfection of Flag-RINGmut or cotransfection of Flag-gp78 with ubiquitin mutated at three critical lysine residues (K29, 48, 63R) involved in polyubiquitin chain elongation. Short hairpin RNA knockdown of Gp78 in HT-1080 fibrosarcoma cells increased mitofusin levels and reduced depolarization-induced mitophagy, whereas siRNA knockdown showed that Mfn1, but not Mfn2, was required for Gp78-dependent depolarization-induced mitophagy. Mitochondrial depolarization induced Gp78-dependent expression of the autophagic marker LC3II and recruitment of enhanced green fluorescent protein-LC3 to the Gp78- and calnexin-labeled, mitochondria-associated ER. Finally, Gp78-induced mitophagy is Parkin independent, as it occurs in Parkin-null HeLa cells and upon siRNA-mediated Parkin knockdown in HEK293 cells. This study therefore describes a novel role for the ER-associated Gp78 ubiquitin ligase and the Mfn1 mitochondrial fusion factor in mitophagy.
PDF
