Integrin α6 signaling induces STAT3-TET3-mediated hydroxymethylation of genes critical for maintenance of glioma stem cells.

Both the extracellular matrix (ECM) and DNA epigenetic regulation are critical for maintaining stem cell phenotype and cancer progression. Whether and how ECM regulates epigenetic alterations to influence cancer stem cells (CSCs) remain to be explored. Here we report that ECM through laminin-integrin α6 upregulates ten-eleven translocation enzyme 3 (TET3) dioxygenase. TET3 in turn mediates DNA cytosine 5'-hydroxymethylation (5hmC) and upregulates genes critical for maintenance of glioma stem cells (GSCs). Activating integrin α6-FAK pathway increases STAT3 activity, TET3 expression and 5hmC levels in GSCs. Moreover, targeting STAT3 disrupts integrin α6-FAK signaling and inhibits TET3+ GSC maturation in vivo. STAT3 directly regulates TET3 expression and the two proteins are co-localized with 5hmC in GSC clusters. 5hmC is upregulated by STAT3 at the promoters of several tumorigenic genes, including c-Myc, known to be critical for GSCs. In vivo silencing of TET3 in GSC-enriched tumors reduces 5hmC accumulation and expression of the GSC critical genes, leading to tumor growth inhibition. TET3 expression and 5hmC accumulation also co-segregate with integrin α6 in patient malignant glioma. Thus, ECM- integrin α6-STAT3-TET3 axis regulates hydroxymethylation of genes important for GSCs, thereby increasing GSC tumorigenicity and resistance to therapies.

Development of a Diagnostic Programmed Cell Death 1-Ligand 1 Immunohistochemistry Assay for Nivolumab Therapy in Melanoma.

Nivolumab is a monoclonal antibody that blocks the interaction between programmed cell death 1 (PD1) and programmed cell death 1-ligand 1 (PD-L1), resulting in enhanced antitumor activity by the immune system. Nivolumab is currently approved by the US Food and Drug Administration (FDA) for melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, and urothelial carcinoma. PD-L1 IHC 28-8 pharmDx is FDA-approved as a complementary diagnostic for immunohistochemical (IHC) detection of PD-L1 in non-squamous NSCLC and melanoma. We report validation of PD-L1 IHC 28-8 pharmDx for PD-L1 detection on formalin-fixed, paraffin-embedded human melanoma specimens using Autostainer Link 48. A prevalence assessment of 104 melanoma specimens indicated that PD-L1 was detected across the full expression level range (0% to 100% of tumor cells). Assay robustness and precision studies were conducted at Agilent Technologies, with additional reproducibility studies performed at 3 external laboratories. Precision studies evaluated at ≥1% and ≥5% expression levels revealed a range of average negative agreement from 89.5%, 95% CI (83.2, 93.6) to 100%, 95% CI (97.3, 100), and average positive agreement from 85.5%, 95% CI (77.6, 90.9) to 100%, 95% CI (97.9, 100). For external reproducibility, precise results were obtained. These results demonstrate PD-L1 IHC 28-8 pharmDx is a precise, robust, and reproducible assay for determining PD-L1 expression in melanoma. This is the first PD-L1 IHC test to receive FDA approval as a complementary diagnostic in melanoma patients whereby positive PD-L1 expression is correlated with the magnitude of nivolumab treatment effect.

Sphingosine-1-Phosphate Receptor-1 Promotes Environment-Mediated and Acquired Chemoresistance.

Drug resistance is a major barrier for the development of effective and durable cancer therapies. Overcoming this challenge requires further defining the cellular and molecular mechanisms underlying drug resistance, both acquired and environment-mediated drug resistance (EMDR). Here, using neuroblastoma (NB), a childhood cancer with high incidence of recurrence due to resistance to chemotherapy, as a model we show that human bone marrow-mesenchymal stromal cells induce tumor expression of sphingosine-1-phosphate receptor-1 (S1PR1), leading to their resistance to chemotherapy. Targeting S1PR1 by shRNA markedly enhances etoposide-induced apoptosis in NB cells and abrogates EMDR, while overexpression of S1PR1 significantly protects NB cells from multidrug-induced apoptosis via activating JAK-STAT3 signaling. Elevated S1PR1 expression and STAT3 activation are also observed in human NB cells with acquired resistance to etoposide. We show in vitro and in human NB xenograft models that treatment with FTY720, an FDA-approved drug and antagonist of S1PR1, dramatically sensitizes drug-resistant cells to etoposide. In summary, we identify S1PR1 as a critical target for reducing both EMDR and acquired chemoresistance in NB. Mol Cancer Ther; 16(11); 2516-27. ©2017 AACR.

SLC7A11 Overexpression in Glioblastoma Is Associated with Increased Cancer Stem Cell-Like Properties.

System xc- is a sodium-independent electroneutral transporter, comprising a catalytic subunit xCT (SLC7A11), which is involved in importing cystine. Certain cancers such as gliomas upregulate the expression of system xc-, which confers a survival advantage against the detrimental effects of reactive oxygen species (ROS) by increasing generation of the antioxidant glutathione. However, ROS have also been shown to function as targeted, intracellular second messengers in an array of physiological processes such as proliferation. Several studies have implicated ROS in important cancer features such as migration, invasion, and contribution to a cancer stem cell (CSC)-like phenotype. The role of system xc- in regulating these ROS-sensitive processes in glioblastoma multiforme (GBM), the most aggressive malignant primary brain tumor in adults, remains unknown. Stable SLC7A11 knockdown and overexpressing U251 glioma cells were generated and characterized to understand the role of redox and system xc- in glioma progression. SLC7A11 knockdown resulted in higher endogenous ROS levels and enhanced invasive properties. On the contrary, overexpression of SLC7A11 resulted in decreased endogenous ROS levels as well as decreased migration and invasion. However, SLC7A11-overexpressing cells displayed actin cytoskeleton changes reminiscent of epithelial-like cells and exhibited an increased CSC-like phenotype. The enhanced CSC-like phenotype may contribute to increased chemoresistance and suggests that overexpression of SLC7A11 in the context of GBM may contribute to tumor progression. These findings have important implications for cancer management where targeting system xC- in combination with other chemotherapeutics can reduce cancer resistance and recurrence and improve GBM patient survival.

Increased Expression of System xc- in Glioblastoma Confers an Altered Metabolic State and Temozolomide Resistance.

Glioblastoma multiforme is the most aggressive malignant primary brain tumor in adults. Several studies have shown that glioma cells upregulate the expression of xCT (SLC7A11), the catalytic subunit of system xc-, a transporter involved in cystine import, that modulates glutathione production and glioma growth. However, the role of system xc- in regulating the sensitivity of glioma cells to chemotherapy is currently debated. Inhibiting system xc- with sulfasalazine decreased glioma growth and survival via redox modulation, and use of the chemotherapeutic agent temozolomide together with sulfasalazine had a synergistic effect on cell killing. To better understand the functional consequences of system xc- in glioma, stable SLC7A11-knockdown and -overexpressing U251 glioma cells were generated. Modulation of SLC7A11 did not alter cellar proliferation but overexpression did increase anchorage-independent cell growth. Knockdown of SLC7A11 increased basal reactive oxygen species (ROS) and decreased glutathione generation resulting in increased cell death under oxidative and genotoxic stress. Overexpression of SLC7A11 resulted in increased resistance to oxidative stress and decreased chemosensitivity to temozolomide. In addition, SLC7A11 overexpression was associated with altered cellular metabolism including increased mitochondrial biogenesis, oxidative phosphorylation, and ATP generation. These results suggest that expression of SLC7A11 in the context of glioma contributes to tumorigenesis, tumor progression, and resistance to standard chemotherapy. IMPLICATIONS: SLC7A11, in addition to redox modulation, appears to be associated with increased cellular metabolism and is a mediator of temozolomide resistance in human glioma, thus making system xC- a potential therapeutic target in glioblastoma multiforme. Mol Cancer Res; 14(12); 1229-42. ©2016 AACR.

Current methods of epitope identification for cancer vaccine design.

The importance of the immune system in tumor development and progression has been emerging in many cancers. Previous cancer vaccines have not shown long-term clinical benefit possibly because were not designed to avoid eliciting regulatory T-cell responses that inhibit the anti-tumor immune response. This review will examine different methods of identifying epitopes derived from tumor associated antigens suitable for immunization and the steps used to design and validate peptide epitopes to improve efficacy of anti-tumor peptide-based vaccines. Focusing on in silico prediction algorithms, we survey the advantages and disadvantages of current cancer vaccine prediction tools.

TLR9 is critical for glioma stem cell maintenance and targeting.

Understanding supports for cancer stem-like cells in malignant glioma may suggest therapeutic strategies for their elimination. Here, we show that the Toll-like receptor TLR9 is elevated in glioma stem-like cells (GSC) in which it contributes to glioma growth. TLR9 overexpression is regulated by STAT3, which is required for GSC maintenance. Stimulation of TLR9 with a CpG ligand (CpG ODN) promoted GSC growth, whereas silencing TLR9 expression abrogated GSC development. CpG-ODN treatment induced Frizzled4-dependent activation of JAK2, thereby activating STAT3. Targeted delivery of siRNA into GSC was achieved via TLR9 using CpG-siRNA conjugates. Through local or systemic treatment, administration of CpG-Stat3 siRNA to silence STAT3 in vivo reduced GSC along with glioma growth. Our findings identify TLR9 as a functional marker for GSC and a target for the delivery of efficacious therapeutics for glioma treatment. Cancer Res; 74(18); 5218-28. ©2014 AACR.

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells.

Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte-associated antigen 4 (CTLA4(apt)) allows gene silencing in exhausted CD8⁺ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8⁺ T cells in the tumor milieu; therefore, CTLA4(apt) fused to a STAT3-targeting siRNA (CTLA4(apt)-STAT3 siRNA) resulted in internalization into tumor-associated CD8⁺ T cells and silencing of STAT3, which activated tumor antigen-specific T cells in murine models. Both local and systemic administration of CTLA4(apt)-STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4(apt)-STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4(apt)-STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4(apt)-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis.

Loss of androgen receptor expression promotes a stem-like cell phenotype in prostate cancer through STAT3 signaling.

Androgen receptor (AR) signaling is important for prostate cancer progression. However, androgen-deprivation and/or AR targeting-based therapies often lead to resistance. Here, we demonstrate that loss of AR expression results in STAT3 activation in prostate cancer cells. AR downregulation further leads to development of prostate cancer stem-like cells (CSC), which requires STAT3. In human prostate tumor tissues, elevated cancer stem-like cell markers coincide with those cells exhibiting high STAT3 activity and low AR expression. AR downregulation-induced STAT3 activation is mediated through increased interleukin (IL)-6 expression. Treating mice with soluble IL-6 receptor fusion protein or silencing STAT3 in tumor cells significantly reduced prostate tumor growth and CSCs. Together, these findings indicate an opposing role of AR and STAT3 in prostate CSC development.

Regulation of adipose tissue T cell subsets by Stat3 is crucial for diet-induced obesity and insulin resistance.

Dysregulated inflammation in adipose tissue, marked by increased proinflammatory T-cell accumulation and reduced regulatory T cells (Tregs), contributes to obesity-associated insulin resistance. The molecular mechanisms underlying T-cell-mediated inflammation in adipose tissue remain largely unknown, however. Here we show a crucial role for signal transducer and activator of transcription 3 (Stat3) in T cells in skewing adaptive immunity in visceral adipose tissue (VAT), thereby contributing to diet-induced obesity (DIO) and insulin resistance. Stat3 activity is elevated in obese VAT and in VAT-resident T cells. Functional ablation of Stat3 in T cells reduces DIO, improves insulin sensitivity and glucose tolerance, and suppresses VAT inflammation. Importantly, Stat3 ablation reverses the high Th1/Treg ratio in VAT of DIO mice that is likely secondary to elevated IL-6 production, leading in turn to suppression of Tregs. In addition, Stat3 in T cells in DIO mice affects adipose tissue macrophage accumulation and M2 phenotype. Our study identifies Stat3 in VAT-resident T cells as an important mediator and direct target for regulating adipose tissue inflammation, DIO, and its associated metabolic dysfunctions.

Evidence that cathelicidin peptide LL-37 may act as a functional ligand for CXCR2 on human neutrophils.

LL-37, derived from human cathelicidin, stimulates immune responses in neutrophils. Although FPR2 and P2X7 were proposed as LL-37 receptors, we have shown that among 21 neutrophil receptors only CXCR2 was down-regulated by LL-37. LL-37 functions similarly to CXCR2-specific chemokines CXCL1 and CXCL7 in terms of receptor down-regulation and intracellular calcium mobilization on freshly isolated neutrophils. Neutrophils pretreated with CXCL8, a chemokine that binds both CXCR1/2, completely blocked the calcium mobilization in response to LL-37, while LL-37 also partially inhibited (125)I-CXCL8 binding to neutrophils. SB225002, a selective CXCR2 antagonist, blocked LL-37-induced calcium mobilization and migration of neutrophils. LL-37 stimulates calcium mobilization in CXCR2-transfected HEK293 cells, CXCR2(+) THP-1 cells and monocytes, but not in CXCR1-transfected HEK293 cells. WKYMVm peptide (ligand for FPR2) does not block LL-37-stimulated calcium flux in either THP-1 (FPR2(-)) or monocytes (FPR2(high)), further confirming the specificity of LL-37 for CXCR2 and not FPR2. Among all ligands tested (ATP, BzATP, WKYMVm, CXCL1, and LL-37), only LL-37 stimulated migration of monocytes (CXCR2(+) and FPR2(+)) and migration was inhibited by the CXCR2 inhibitor SB225002. Moreover, CXCR2 but not CXCR1 was internalized in LL-37-treated neutrophils. Thus, our data provide evidence that LL-37 may act as a functional ligand for CXCR2 on human neutrophils.

Neutrophil secondary necrosis is induced by LL-37 derived from cathelicidin.

Neutrophils represent the most common granulocyte subtype present in blood. The short half-life of circulating neutrophils is regulated by spontaneous apoptosis, and tissue infiltrating neutrophils die by apoptosis and secondary necrosis. The mechanism of neutrophil apoptosis has been the subject of many studies; however, the mechanism of neutrophil secondary necrosis is less clear. Human cathelicidin cationic peptide 18, proteolytically processed to its active form, LL-37, is secreted by neutrophils and epithelial cells and shown to have effects in addition to bacterial lysis. We demonstrate here that LL-37 affects neutrophil lifespan by the pathway of secondary necrosis, rapidly converting annexin V-positive (AV(+)), propidium iodide-negative (PI(-); apoptotic) cells into PI(+) (necrotic) cells with the release of IL-8, IL-1R antagonist, ATP, and intact granules. The effects of LL-37 on apoptotic neutrophils are neither energy-dependent nor affected by pretreatment with G-CSF, GM-CSF, TNF-alpha, and LPS and are partially inhibited by human serum. Moreover, LL-37 decreases CXCR2 expression of AV(-)PI(-) (live) neutrophils, suggesting an effect on the neutrophil response to its chemotactic factors, including IL-8. Thus, the lifespan and inflammatory functions of neutrophils are directly affected by LL-37.

High plasma levels of MCP-1 and eotaxin provide evidence for an immunological basis of fibromyalgia.

Fibromyalgia (FMS), a predominantly female (85%) syndrome, affects an estimated 2% of the US population with skeletal muscle ache, fatigue, headache, and sleep disorder. The pathogenesis of FMS is unknown and there is no laboratory test for diagnosis. In this study, plasma levels of 25 cytokines and chemokines in 92 female patients with FMS and 69 family members were measured compared to 77 controls. Trans-endothelial migration of normal leukocytes in response to FMS plasma and the cytokine profile of human myoblasts were analyzed. High levels of MCP-1 (P<0.001) and eotaxin (P<0.01) were found in patients and family members compared to controls. Patients (56/92) treated with the single agent guaifenesin (>3 months) had higher levels of eotaxin than those not treated (P<0.01). Diluted plasma from patients increased the migration of normal eosinophils and monocytes, but not neutrophils, through an endothelial/Matrigel barrier only when mast cells are included in the lower wells (P<0.05). Furthermore, myoblasts can secrete MCP-1, eotaxin, and IP-10, while treatment with MCP-1 caused secretion of IL-1beta, eotaxin and IP-10. FMS is associated with inflammatory chemokines, that MCP-1 and eotaxin may contribute to the symptoms of FMS, and that similar cytokine profiles found in family members support the idea that FMS has a genetic component. Furthermore, the chemokine profile associated with FMS has direct effects on the migration of eosinophils and monocytes in the presence of mast cells, and skeletal muscle itself may secrete.