Biodistribution and Activity of EGFR Targeted Polymeric Micelles Delivering a New Inhibitor of DNA Repair to Orthotopic Colorectal Cancer Xenografts with Metastasis.

The disruption of polynucleotide kinase/phosphatase (PNKP) in colorectal cancer (CRC) cells deficient in phosphatase and tensin homolog (PTEN) is expected to lead to the loss of cell viability by a process known as synthetic lethality. In previous studies, we have reported on the encapsulation of a novel inhibitor of PNKP, namely, A83B4C63, in polymeric micelles and its activity in slowing the growth of PTEN-deficient CRC cells as well as subcutaneous xenografts. In this study, to enhance drug delivery and specificity to CRC tumors, the surface of polymeric micelles carrying A83B4C63 was modified with GE11, a peptide targeting epidermal growth factor receptor (EGFR) overexpressed in about 70% of CRC tumors. Using molecular dynamics (MD) simulations, we assessed the binding site and affinity of GE11 for EGFR. The GE11-modified micelles, tagged with a near-infrared fluorophore, showed enhanced internalization by EGFR-overexpressing CRC cells in vitro and a trend toward increased primary tumor homing in an orthotopic CRC xenograft in vivo. In line with these observations, the GE11 modification of polymeric micelles was shown to positively contribute to the improved therapeutic activity of encapsulated A83B4C63 against HCT116-PTEN-/- cells in vitro and that of orthotopic CRC xenograft in vivo. In conclusion, our results provided proof of principle evidence for the potential benefit of EGFR targeted polymeric micellar formulations of A83B4C63 as monotherapeutics for aggressive and metastatic CRC tumors but at the same time highlighted the need for the development of EGFR ligands with improved physiological stability and EGFR binding.

Intratumoral STING activation causes durable immunogenic tumor eradication in the KP soft tissue sarcoma model.

Introduction: Soft tissue sarcomas (STS) are highly metastatic, connective-tissue lineage solid cancers. Immunologically, sarcomas are frequently characterized by a paucity of tumor infiltrating lymphocytes and an immune suppressive microenvironment. Activation of the STING pathway can induce potent immune-driven anti-tumor responses within immunogenic solid tumors; however, this strategy has not been evaluated in immunologically cold sarcomas. Herein, we assessed the therapeutic response of intratumoral STING activation in an immunologically cold murine model of undifferentiated pleomorphic sarcoma (UPS). Materials and Results: A single intratumoral injection of the murine STING agonist, DMXAA resulted in durable cure in up to 60% of UPS-bearing mice. In mice with synchronous lung metastases, STING activation within hindlimb tumors resulted in 50% cure in both anatomic sites. Surviving mice all rejected UPS re-challenge in the hindlimb and lung. Therapeutic efficacy of STING was inhibited by lymphocyte deficiency but unaffected by macrophage deficiency. Immune phenotyping demonstrated enrichment of lymphocytic responses in tumors at multiple timepoints following treatment. Immune checkpoint blockade enhanced survival following STING activation. Discussion: These data suggest intratumoral activation of the STING pathway elicits local and systemic anti-tumor immune responses in a lymphocyte poor sarcoma model and deserves further evaluation as an adjunctive local and systemic treatment for sarcomas.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade.

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-'quiescent' microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

A synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3'-phosphatase (PNKP) for targeted therapy of PTEN-deficient colorectal cancer.

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN-/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.

Nano-Delivery of a Novel Inhibitor of Polynucleotide Kinase/Phosphatase (PNKP) for Targeted Sensitization of Colorectal Cancer to Radiation-Induced DNA Damage.

Inhibition of the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) increases the sensitivity of cancer cells to DNA damage by ionizing radiation (IR). We have developed a novel inhibitor of PNKP, i.e., A83B4C63, as a potential radio-sensitizer for the treatment of solid tumors. Systemic delivery of A83B4C63, however, may sensitize both cancer and normal cells to DNA damaging therapeutics. Preferential delivery of A83B4C63 to solid tumors by nanoparticles (NP) was proposed to reduce potential side effects of this PNKP inhibitor to normal tissue, particularly when combined with DNA damaging therapies. Here, we investigated the radio-sensitizing activity of A83B4C63 encapsulated in NPs (NP/A83) based on methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) or solubilized with the aid of Cremophor EL: Ethanol (CE/A83) in human HCT116 colorectal cancer (CRC) models. Levels of γ-H2AX were measured and the biodistribution of CE/A83 and NP/A83 administered intravenously was determined in subcutaneous HCT116 CRC xenografts. The radio-sensitization effect of A83B4C63 was measured following fractionated tumor irradiation using an image-guided Small Animal Radiation Research Platform (SARRP), with 24 h pre-administration of CE/A83 and NP/A83 to Luc+/HCT116 bearing mice. Therapeutic effects were analyzed by monitoring tumor growth and functional imaging using Positron Emission Tomography (PET) and [18F]-fluoro-3'-deoxy-3'-L:-fluorothymidine ([18F]FLT) as a radiotracer for cell proliferation. The results showed an increased persistence of DNA damage in cells treated with a combination of CE/A83 or NP/A83 and IR compared to those only exposed to IR. Significantly higher tumor growth delay in mice treated with a combination of IR and NP/A83 than those treated with IR plus CE/A83 was observed. [18F]FLT PET displayed significant functional changes for tumor proliferation for the drug-loaded NP. This observation was attributed to the higher A83B4C63 levels in the tumors for NP/A83-treated mice compared to those treated with CE/A83. Overall, the results demonstrated a potential for A83B4C63-loaded NP as a novel radio-sensitizer for the treatment of CRC.

Is Use of BMP-2 Associated with Tumor Growth and Osteoblastic Differentiation in Murine Models of Osteosarcoma?

BACKGROUND: The putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent. QUESTIONS/PURPOSES: (1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma. METHODS: In this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H2O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation. RESULTS: Constitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm versus 1316 ± 387.4 mm, mean difference 1009 mm [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm versus 326.1 ± 72.8 mm, mean difference 326.1 mm [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10 ± 8.3x10 photons/s versus 9.3 x 10 ± 1.5 x 10 photons/s, mean difference 8.6 x 10 photons/s [95% CI 5.1 x 10 to 1.2 x 10]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm versus 1335 ± 102.7 mm, mean difference 840.3 mm [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions. CONCLUSIONS: As demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted. CLINICAL RELEVANCE: Our results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma.

Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis.

The cysteine protease inhibitor Cystatin C (CST3) is highly expressed in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental autoimmune encephalomyelitis (EAE; a model of MS), but its roles in the diseases are unknown. Here, we show that CST3 plays a detrimental function in myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE but only in female animals. Female Cst3 null mice display significantly lower clinical signs of disease compared to wild-type (WT) littermates. This difference is associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, major histocompatibility complex [MHC] II, LC3A/B) involved in antigen processing, presentation, and co-stimulation in antigen-presenting cells (APCs). In contrast, male WT and Cst3-/- mice and cells show no differences in EAE signs or APC function. Further, the sex-dependent effect of CST3 in EAE is sensitive to gonadal hormones. Altogether, we have shown that CST3 has a sex-dependent role in MOG35-55-induced EAE.

Simple and robust polymer-based sensor for rapid cancer detection using serum.

We report a polymer-based sensor that rapidly detects cancer based on changes in serum protein levels. Using three ratiometric fluorescence outputs, this simple system identifies early stage and metastatic lung cancer with a high level of accuracy exceeding many biomarker-based assays, making it an attractive strategy for point-of-care testing.

Recombinant human PRG4 (rhPRG4) suppresses breast cancer cell invasion by inhibiting TGFß-Hyaluronan-CD44 signalling pathway.

Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFß) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFß-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFß-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFß has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFß has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFß actions. Finally, these findings point to rhPRG4's potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.

Expression of a constitutively active human STING mutant in hematopoietic cells produces an Ifnar1-dependent vasculopathy in mice.

STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disorder characterized by blood vessel occlusions, acral necrosis, myositis, rashes, and pulmonary inflammation that are the result of activating mutations in the STimulator of Interferon Genes (STING). We generated a transgenic line that recapitulates many of the phenotypic aspects of SAVI by targeting the expression of the human STING-N154S-mutant protein to the murine hematopoietic compartment. hSTING-N154S mice demonstrated failure to gain weight, lymphopenia, progressive paw swelling accompanied by inflammatory infiltrates, severe myositis, and ear and tail necrosis. However, no significant lung inflammation was observed. X-ray microscopy imaging revealed vasculopathy characterized by arteriole occlusions and venous thromboses. Type I interferons and proinflammatory mediators were elevated in hSTING-N154S sera. Importantly, the phenotype was prevented in hSTING-N154S mice lacking the type I interferon receptor gene (Ifnar1). This model, based on a mutant human STING protein, may shed light on the pathophysiological mechanisms operative in SAVI.

Ratiometric Array of Conjugated Polymers-Fluorescent Protein Provides a Robust Mammalian Cell Sensor.

Supramolecular complexes of a family of positively charged conjugated polymers (CPs) and green fluorescent protein (GFP) create a fluorescence resonance energy transfer (FRET)-based ratiometric biosensor array. Selective multivalent interactions of the CPs with mammalian cell surfaces caused differential change in FRET signals, providing a fingerprint signature for each cell type. The resulting fluorescence signatures allowed the identification of 16 different cell types and discrimination between healthy, cancerous, and metastatic cells, with the same genetic background. While the CP-GFP sensor array completely differentiated between the cell types, only partial classification was achieved for the CPs alone, validating the effectiveness of the ratiometric sensor. The utility of the biosensor was further demonstrated in the detection of blinded unknown samples, where 121 of 128 samples were correctly identified. Notably, this selectivity-based sensor stratified diverse cell types in minutes, using only 2000 cells, without requiring specific biomarkers or cell labeling.

Characterization of a murine model of metastatic human non-small cell lung cancer and effect of CXCR4 inhibition on the growth of metastases.

Despite successful preclinical testing carried out through the use of subcutaneous xenografted tumors, many anti-cancer agents have gone on to fail in human trials. One potential factor accounting for this discrepancy may relate to the inadequacy of the commonly employed preclinical models to recapitulate the human disease, particularly when it comes to discovery of agents that are effective against advanced disease. Herein, we report the characterization of a NSCLC model and an exploration of the impact that a CXCR4 inhibitor, AMD3100, had on NCI-H1299-derived metastasis. These cells express a variety of metastasis-promoting factors, hence we selected them for a study of their metastatic colonization potential. To accomplish this, luciferase-expressing H1299 (H1299-luc2) cells were inoculated into athymic mice via the intracardiac route. This strategy produced adrenal, bone, ovarian, and pancreatic metastases, sites commonly involved in human metastatic NSCLC. Notably, micro-computed tomography and histological evaluation of the skeletal lesions revealed the presence of extensive osteolysis. To investigate the potential role of CXCR4 in mediating metastatic colonization of tissues, AMD3100 was administered to mice inoculated with H1299-luc2 cells. While this treatment did not appreciably alter the frequency of metastatic colonization, it was able to slow the growth of macrometastases. This model, recapitulating some of the events seen in late-stage human NSCLC, may prove useful in the evaluation of new therapies targeting metastatic disease.

DNA mismatch repair deficiency accelerates lung neoplasm development in K-ras(LA1/+) mice: a brief report.

Inherited as well as acquired deficiencies in specific DNA mismatch repair (MMR) components are associated with the development of a wide range of benign and malignant neoplasms. Loss of key members such as MSH2 and MLH1 severely cripples the ability of the cell to recognize and correct such lesions as base:base mismatches and replicative DNA polymerase errors such as slippages at repetitive sequences. Genomic instability resulting from MMR deficiency not only predisposes cells to malignant transformation but may also promote tumor progression. To test the latter, we interbred Msh2(-/-) mice with the K-ras(LA1/+) transgenic line that spontaneously develops a range of premalignant and malignant lung lesions. Compared to K-ras(LA1/+) mice, K-ras(LA1/+); Msh2(-/-) mice developed lung adenomas and adenocarcinomas at an increased frequency and also demonstrated evidence of accelerated adenocarcinoma growth. Since MMR defects have been identified in some human lung cancers, the mutant mice may not only be of preclinical utility but they will also be useful in identifying gene alterations able to act in concert with Kras mutants to promote tumor progression.

Mind the gap: genetic manipulation of basicranial growth within synchondroses modulates calvarial and facial shape in mice through epigenetic interactions.

Phenotypic integration patterns in the mammalian skull have long been a focus of intense interest as a result of their suspected influence on the trajectory of hominid evolution. Here we test the hypothesis that perturbation of cartilage growth, which directly affects only the chondrocranium during development, will produce coordinated shape changes in the adult calvarium and face regardless of mechanism. Using two murine models of cartilage undergrowth that target two very different mechanisms, we show that strong reduction in cartilage growth produces a short, wide, and more flexed cranial base. This in turn produces a short, wide face in both models. Cranial base and face are already correlated early in ontogeny, and the relationship between these modules gains structure through postnatal growth and development. These results provide further evidence that there exist physical interactions between developing parts of the phenotype that produce variation at a distance from the actual locus upon which a particular selective pressure is acting. Phenotypic changes observed over the course of evolution may not all require adaptationist explanations; rather, it is likely that a substantial portion of observed phenotypic variation over the history of a clade is not directly adaptive but rather a secondary consequence of some local response to selection.

Combined treatment with paclitaxel and suramin prevents the development of metastasis by inhibiting metastatic colonization of circulating tumor cells.

Metastatic disease accounts for most deaths due to breast cancer and thus identification of novel ways to prevent this complication remains a key goal. A frequently employed preclinical model of breast cancer metastasis relies on xenografted human MDA-MB-231 cells, since these reliably produce both soft tissue and osseous metastases when introduced into the arterial circulation of athymic mice. Herein, we explored the ability of suramin (SA), an agent shown to antagonize the effects of various stromal cell-derived growth factors relevant to bone marrow colonization of tumor cells, administered both with and without paclitaxel (PTX), to inhibit the development of MDA-MB-231 metastases. Treatment with SA, PTX, or PTX plus SA (PTX/SA) was begun either at day-1, or 7 days after intra-arterial inoculation of luciferase-expressing MDA-MB-231-luc2 cells. Using in vivo and ex vivo bioluminescence imaging to detect macro-metastases, we found that PTX/SA treatment initiated on day-1 was able to dramatically reduce the frequency of bone metastases. PTX/SA and PTX administration commenced at day 7, in contrast, had no significant effect on the frequency of bone metastases, but exerted a relatively modest inhibitory effect on growth of metastases. Interestingly, reminiscent of what is seen clinically in anti-HER2 treated individuals, several of the PTX/SA-treated long term survivors went on to develop late onset CNS metastasis. Our results suggest that combining SA with PTX either in an adjuvant setting or during medical interventions that can increase the numbers of circulating tumour cells might be an effective way to prevent the development of metastases.

DMXAA causes tumor site-specific vascular disruption in murine non-small cell lung cancer, and like the endogenous non-canonical cyclic dinucleotide STING agonist, 2'3'-cGAMP, induces M2 macrophage repolarization.

The vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a murine agonist of the stimulator of interferon genes (STING), appears to target the tumor vasculature primarily as a result of stimulating pro-inflammatory cytokine production from tumor-associated macrophages (TAMs). Since there were relatively few reports of DMXAA effects in genetically-engineered mutant mice (GEMM), and models of non-small cell lung cancer (NSCLC) in particular, we examined both the effectiveness and macrophage dependence of DMXAA in various NSCLC models. The DMXAA responses of primary adenocarcinomas in K-rasLA1/+ transgenic mice, as well as syngeneic subcutaneous and metastatic tumors, generated by a p53R172HΔg/+; K-rasLA1/+ NSCLC line (344SQ-ELuc), were assessed both by in vivo bioluminescence imaging as well as by histopathology. Macrophage-dependence of DMXAA effects was explored by clodronate liposome-mediated TAM depletion. Furthermore, a comparison of the vascular structure between subcutaneous tumors and metastases was carried out using micro-computed tomography (micro-CT). Interestingly, in contrast to the characteristic hemorrhagic necrosis produced by DMXAA in 344SQ-ELuc subcutaneous tumors, this agent failed to cause hemorrhagic necrosis of either 344SQ-ELuc-derived metastases or autochthonous K-rasLA1/+ NSCLCs. In addition, we found that clodronate liposome-mediated depletion of TAMs in 344SQ-ELuc subcutaneous tumors led to non-hemorrhagic necrosis due to tumor feeding-vessel occlusion. Since NSCLC were comprised exclusively of TAMs with anti-inflammatory M2-like phenotype, the ability of DMXAA to re-educate M2-polarized macrophages was examined. Using various macrophage phenotypic markers, we found that the STING agonists, DMXAA and the non-canonical endogenous cyclic dinucleotide, 2'3'-cGAMP, were both capable of re-educating M2 cells towards an M1 phenotype. Our findings demonstrate that the choice of preclinical model and the anatomical site of a tumor can determine the vascular disrupting effectiveness of DMXAA, and they also support the idea of STING agonists having therapeutic utility as TAM repolarizing agents.

NADPH oxidase modifies patterns of MHC class II-restricted epitopic repertoires through redox control of antigen processing.

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow-derived macrophages, but not dendritic cells, to process and present the I-A(b)-immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-A(b) binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-A(b) mice that were deficient in the p47(phox) or gp91(phox) subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4(+) T cells in the CNS, despite eliciting a normal primary CD4(+) T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4(+) T cell-driven autoimmune disease processes.

Prostate epithelium-specific deletion of the selenocysteine tRNA gene Trsp leads to early onset intraepithelial neoplasia.

Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.

A novel role for the SUMO E3 ligase PIAS1 in cancer metastasis.

Tumor metastasis contributes to the grave morbidity and mortality of cancer, but the mechanisms underlying tumor cell invasiveness and metastasis remain incompletely understood. Here, we report that expression of the SUMO E3 ligase PIAS1 suppresses TGFß-induced activation of the matrix metalloproteinase MMP2 in human breast cancer cells. We also find that knockdown of endogenous PIAS1 or inhibition of its SUMO E3 ligase activity stimulates the ability of TGFß to induce an aggressive phenotype in three-dimensional breast cancer cell organoids. Importantly, inhibition of the SUMO E3-ligase activity of PIAS1 in breast cancer cells promotes metastases in mice in vivo. Collectively, our findings define a novel and critical role for the SUMO E3 ligase PIAS1 in the regulation of the invasive and metastatic potential of malignant breast cancer cells. These findings advance our understanding of cancer invasiveness and metastasis with potential implications for the development of biomarkers and therapies in breast cancer.

Targeted cancer therapeutics: biosynthetic and energetic pathways characterized by metabolomics and the interplay with key cancer regulatory factors.

Reprogramming of energy metabolism has recently been added to the list of hallmarks that define cancer. Cellular metabolism plays a central role in cancer initiation and progression to metastatic disease. Genotypic and phenotypic metabolic alterations are seen throughout tumourigenesis, allowing cancer cells to sustain increased rates of proliferation. Furthermore, this shift fuels necessary substrates for nucleotide, protein, and lipid synthesis to support cell growth. Beyond the 'Warburg effect', the widely observed increase in the glycolytic processing of glucose in cancer cells, numerous other metabolic changes have been characterized in cancer. Metabolomics provides a valuable platform for the investigation of the metabolic perturbations that occur in different disease states using a systems biology approach to determine metabolic profiles of biological samples. As cell metabolism is a complex network of interdependent pathways, local alterations will have an impact on overall tumor metabolism. In this review, we will highlight particular pathways, including glycolysis, nucleotide biosynthesis, lipid metabolism, and bioenergetics with an eye towards selected metabolic targets that may provide a novel approach to therapeutic development. Specific regulatory factors, including Myc, p53, HIF-1 and mTOR are briefly highlighted, as well as the key signaling pathways that can affect cellular metabolism. To demonstrate the powerful utility of high-throughput metabolite profiling techniques, we present a practical example of the metabolomic profiling of metastatic cells derived from a lung cancer metastasis model.