Oliver Glass

HER2 Overexpression Elicits a Proinflammatory IL-6 Autocrine Signaling Loop That Is Critical for Tumorigenesis

HER2 overexpression occurs in approximately 25% of breast cancers, where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis, although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of interleukin-6 (IL-6) expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2-amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL-6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers.

Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity

Recombinant serotype 5 adenovirus (Ad5) vectors lacking E1 expression induce robust immune responses against encoded transgenes in pre-clinical models, but have muted responses in human trials because of widespread pre-existing anti-adenovirus immunity. Attempts to circumvent Ad5-specific immunity by using alternative serotypes or modifying capsid components have not yielded profound clinical improvement. To address this issue, we explored a novel alternative strategy, specifically reducing the expression of structural Ad5 genes by creating E1 and E2b deleted recombinant Ad5 vectors. Our data show that [E1−, E2b−]vectors retaining the Ad5 serotype are potent immunogens in pre-clinical models despite the presence of significant Ad5-specific immunity, in contrast to [E1−] vectors. These pre-clinical studies with E1 and E2b-deleted recombinant Ad5 vectors suggest that anti-Ad immunity will no longer be a limiting factor, and that clinical trials to evaluate their performance are warranted.

Ligand-Independent Toll-like Receptor Signals Generated by Ectopic Overexpression of MyD88 Generate Local and Systemic Antitumor Immunity

Although critical for initiating and regulating immune responses, the therapeutic use of individual cytokines as anticancer immunotherapeutic agents has achieved only modest clinical success. Consequently, many current strategies have focused on the use of specific immunotherapeutic agonists that engage individual receptors of innate immune networks, such as the Toll-like receptor (TLR) system, each resulting in specific patterns of gene expression, cytokine production, and inflammatory outcome. However, these immunotherapeutics are constrained by variable cellular TLR expression and responsiveness to particular TLR agonists, as well as the specific cellular context of different tumors. We hypothesized that overexpression of MyD88, a pivotal regulator of multiple TLR signaling pathways, could circumvent these constraints and mimic coordinated TLR signaling across all cell types in a ligand-independent fashion. To explore this hypothesis, we generated an adenoviral vector expressing MyD88 and show that Ad-MyD88 infection elicits extensive Th1-specific transcriptional and secreted cytokine signatures in all murine and human cell types tested in vitro and in vivo. Importantly, in vivo intratumoral injection of Ad-MyD88 into established tumor masses enhanced adaptive immune responses and inhibited local tumor immunosuppression, resulting in significantly inhibited local and systemic growth of multiple tumor types. Finally, Ad-MyD88 infection of primary human dendritic cells, tumor-associated fibroblasts, and colorectal carcinoma cells elicited significant Th1-type cytokine responses, resulting in enhanced tumor cell lysis and expansion of human tumor antigen-specific T cells. Thus, Ad-MyD88 initiated robust antitumor activity in established murine tumor microenvironments and in human contexts, suggesting its potential effectiveness as a clinical immunotherapeutic strategy.

Replication-attenuated Human Adenoviral Type 4 vectors elicit capsid dependent enhanced innate immune responses that are partially dependent upon interactions with the complement system

Human Adenovirus Type 4 (HAdV-4) is responsible for epidemic outbreaks of Acute Respiratory Disease (especially in military recruits), and is known to cause significant morbidity with several reported cases of mortality. However, we do not understand why this serotype causes such high morbidity, and have little insight into the immunobiology of HAdV-4 infections. We have now developed a replication attenuated HAdV-4 vector system, and through it, demonstrate that HAdV-4 virions have enhanced infectivity of certain cell types and reveal aspects of the serotype-specific heightened innate immunogenicity of infectious HAdV-4 capsids both in vitro and in vivo. We further found that elements of this serotype-specific immunogenicity were dependent upon interactions with the complement system. These findings provide insights into the mechanisms possibly underlying the known morbidity accompanying wild-type HAdV-4 infections as well as highlight important considerations when considering development of alternative serotype vectors.

An Adenoviral Vaccine Encoding Full-Length Inactivated Human HER2 Exhibits Potent Immunogenicty and Enhanced Therapeutic Efficacy Without Oncogenicity

Purpose: Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in imunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. Experimental Design: We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM), and full-length HER2 inactivated for kinase function (Ad-HER2-ki), and determined their immunogenicity and antitumor effect in wild type (WT) and HER2-tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. Results: Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. Moreover, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage-independent growth, and were not transformed in vivo. Conclusions: Vaccination with mutationally inactivated, nononcogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective antitumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials. Clin Cancer Res; 16(5); 1466–77

Effect of aerobic training on the host systemic milieu in patients with solid tumours: an exploratory correlative study.

Background:Few studies have investigated the effects of exercise on modulation of host factors in cancer patients. We investigated the efficacy of chronic aerobic training on multiple host-related effector pathways in patients with solid tumours.Patients and Methods:Paired peripheral blood samples were obtained from 44 patients with solid tumours receiving cytotoxic therapy and synthetic erythropoietin (usual care; n=21) or usual care plus supervised aerobic training (n=23) for 12 weeks. Samples were characterised for changes in immune, cytokine and angiogenic factors, and metabolic intermediates. Aerobic training consisted of three supervised cycle ergometry sessions per week at 60% to 100% of peak oxygen consumption (VO2peak), 30–45 min per session, for 12 weeks following a nonlinear prescription.Results:The between-group delta change in cardiopulmonary function was +4.1 ml kg −1 min−1, favouring aerobic training (P<0.05). Significant pre–post between-group differences for five cytokine and angiogenic factors (HGF, IL-4, macrophage inflammatory protein-1β (MIP-1β), vascular endothelial growth factor (VEGF), and TNF-α) also favour the aerobic training group (P’s<0.05). These reductions occurred in conjunction with nonsignificant group differences for T lymphocytes CD4+, CD8+, and CD8+/CD45RA (P<0.10). For these factors, circulating concentrations generally increased from baseline to week 12 in the aerobic training group compared with decreases or no change in the usual care group. No significant changes in any metabolic intermediates were observed.Conclusions:Aerobic training alters host availability of select immune–inflammatory effectors in patients with solid tumours; larger confirmatory studies in more homogenous samples are warranted.

Differential response to exercise in claudin-low breast cancer

Exposure to exercise following a breast cancer diagnosis is associated with reductions in the risk of recurrence. However, it is not known whether breast cancers within the same molecular-intrinsic subtype respond differently to exercise. Syngeneic mouse models of claudin-low breast cancer (i.e., EO771, 4TO7, and C3(1)SV40Tag-p16-luc) were allocated to a uniform endurance exercise treatment dose (forced treadmill exercise) or sham-exercise (stationary treadmill). Compared to sham-controls, endurance exercise treatment differentially affected tumor growth rate: 1- slowed (EO771), 2- accelerated (C3(1)SV40Tag-p16-luc), or 3- was not affected (4TO7). Differential sensitivity of the three tumor lines to exercise was paralleled by effects on intratumoral Ki-67, Hif1-α, and metabolic programming. Inhibition of Hif1-α synthesis by the cardiac glycoside, digoxin, completely abrogated exercise-accelerated tumor growth in C3(1)SV40Tag-p16-luc. These results suggest that intratumoral Hif1-α expression is an important determinant of claudin-low breast cancer adaptation to exercise treatment.

Serum Interleukin-8, Osteopontin, and Monocyte Chemoattractant Protein 1 Are Associated With Hepatic Fibrosis in Patients With Nonalcoholic Fatty Liver Disease

The severity of hepatic fibrosis is the primary predictor of liver‐related morbidity and mortality in patients with nonalcoholic fatty liver disease (NAFLD). Unfortunately, noninvasive serum biomarkers for NAFLD‐associated fibrosis are limited. We analyzed baseline serum samples for 24 cytokines of 97 patients with biopsy‐proven NAFLD. These patients were prospectively enrolled in a clinical study (ClinicalTrials.gov NCT00794716) to identify cytokines associated with liver fibrosis in patients with nonalcoholic steatohepatitis. Patients were stratified according to severity of hepatic fibrosis (mild, stage 0‐1, n = 37; moderate, stage 2, n = 40; and advanced, stage 3‐4, n = 20) while controlling for age, race, sex, body mass index, and diabetes mellitus. Interleukin‐8 (IL‐8), osteopontin (OPN), and monocyte chemoattractant protein 1 (MCP1) were associated with liver fibrosis (P < 0.001, P = 0.005, P = 0.016, respectively). After controlling for steatosis, lobular inflammation, hepatocyte ballooning, age, sex, body mass index, diabetes mellitus, hypertension, and metabolic syndrome status, IL‐8 remained strongly associated with fibrosis (P = 0.001). Furthermore, IL‐8 was also a strong predictor of increased fibrotic liver injury compared to established markers of hepatic fibrosis. Hepatic gene expression from 72 patients with NAFLD (n = 40 mild fibrosis; n = 32 advanced fibrosis) from the Duke University Health System NAFLD Clinical Database and Biorepository revealed IL‐8, MCP1, and OPN gene expression to be increased and differentially expressed in patients with advanced hepatic fibrosis. Thus, serum IL‐8, MCP1, and OPN may reflect up‐regulated gene expression during liver fibrosis in NAFLD. Conclusion: Serum IL‐8, MCP1, and OPN may serve as a test for advanced hepatic fibrosis in NAFLD and thus reveal novel targets for antifibrotic therapies. The increased serum IL‐8, MCP1, and OPN that correspond with associated hepatic gene expression lend strength to such analytes as ideal surrogate serum biomarkers for severity of hepatic fibrosis.

Abstract 1376: Exercise alters breast cancer phenotype through distinct reductions in host-derived proinflammatory growth factor ligands.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC It is becoming increasingly recognized that host (systemic) and tumor cell release of proinflammatory growth factors are critical determinants of cancer progression and therapeutic response in patients with solid tumors. Thus, strategies that can attenuate systemic and/or tumor production of proinflammatory growth factors may offer an effective approach to improve therapeutic outcomes following a cancer diagnosis. The present study tests the central hypothesis that exercise modulates systemic levels of key growth factor ligands that, in turn, inhibit the activity of critical downstream cell signaling pathways to effectively inhibit tumor progression. To address this question, we took advantage of sera collected from a clinical trial examining the efficacy of supervised exercise training, relative to sedentary control, in patients with early or advanced solid tumors. Multiplex ELISA analysis showed that exercising patients (N=23) had significant reductions in circulating concentrations of interleukin (IL)-4, MIP1-β (macrophage inflammatory protein-1β), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), and hepatocyte growth factor (HGF) in comparison with patients randomized to sedentary control (N=21). Exposure of estrogen receptor positive (ER+) and Triple-Negative (ER-/PR-/HER2-) distinct human breast cancer cell lines (MCF-7 and MDA-MB-231) to serum from exercised breast cancer patients led to marked alterations in cellular phenotype as shown by increases in proliferation, migration, and apoptosis, compared with exposure to serum from control patients. In vitro ‘add-back’ experiments using recombinant growth factors in concentrations consistent with that observed in the clinical trial, revealed that HGF produced similar alterations in tumor proliferation and apoptosis as that observed with serum from exercising patients. Co-culturing of human breast cancer cells with exercise serum and a neutralizing antibody against HGF, led to increases in proliferation and decreases in apoptosis in comparison to exercise serum alone. These results suggest that growth factor ligand deprivation may play a critical role in mediating the effects of exercise on tumor cellular phenotype. As such, our findings may provide initial insight into the potential mechanisms underlying recent observations showing higher levels of exercise correlate with more favorable disease outcomes in early breast cancer patients. More generally, this study indicates the widespread potential of exercise to modulate growth factor-driven signaling and by extension, tumor progression and possibly innate or acquired resistance to therapy. Citation Format: Oliver Glass, Brant A. Inman, Kerry S. Courneya, John R. Mackey, Erik Nelson, Zachary Hartman, Lee W. Jones. Exercise alters breast cancer phenotype through distinct reductions in host-derived proinflammatory growth factor ligands. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1376. doi:10.1158/1538-7445.AM2013-1376

Abstract 2421: An adenoviral vaccine encoding full-length inactivated human HER2 exhibits potent immunogenicity and enhanced therapeutic efficacy without oncogenicity

While HER2 oncogene directed therapies such as lapatinib and trastuzumab confer clinical benefits, a majority of metastatic HER2+ patients eventually experience progressive disease; however, their tumors remain strongly HER2+. Thus, current therapeutic limitations combined with continued HER2 expression in patients makes immunotherapeutic vaccination against HER2 an attractive strategy. But as HER2 is an oncogene, it is imperative to develop HER2 vaccines with maximal immunologic potential, but minimal oncologic potential. To address these issues, we created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM) and full length HER2 inactivated for kinase function (Ad-HER2-ki) and determined their relative immunogenicity, anti-tumor effectiveness, as well as their oncogenic potential. As an immunotherapeutic vaccine, we found that that both Ad-HER2-ki and Ad-HER2-ECD-TM were highly effective in eliciting significant T-cell and antibody responses to HER2 in naive mouse models compared to plasmid vaccination with HER2-ki constructs, thus validating the immunologic efficacy of the adenoviral platform. In contrast, we did not observe any induction of HER2 specific T-cell or antibody responses in HER2-ECD vaccinated animals. While the strong immune responses from both Ad-HER2-ki and Ad-HER2-ECD-TM vectors translated into significantly retarded tumor growth in naive animals, our studies revealed that Ad-HER2ki vaccinated animals had the most significant HER2-specific T-cell responses as well as the most significant anti-tumor response. When a HER2+ tolerant mouse model was used, we observed that direct Ad-HER2-ki vaccination elicited only slightly diminished T-cell and antibody responses compared to HER2 naive mice. Most significantly, we were able to demonstrate that Ad-HER2-ki vaccination of HER2+ tolerant mice elicits significant anti-HER2+ tumor responses. Subsequent investigation into the oncogenicity associated with strong overexpression of HER2-ki revealed no evidence for its oncogenic functionality in terms of phosphorylation or transcriptional signature in primary human cells. We also found no evidence for its oncogenicity in enabling enhanced cellular proliferation, anchorage-independent growth, or transformation in vivo. In sum, we found that vaccination with mutationally inactivated, non-oncogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translate into strong and effective anti-tumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2421.