Thomas E. Darga

Irradiation and anti–PD-L1 treatment synergistically promote antitumor immunity in mice

High-dose ionizing irradiation (IR) results in direct tumor cell death and augments tumor-specific immunity, which enhances tumor control both locally and distantly. Unfortunately, local relapses often occur following IR treatment, indicating that IR-induced responses are inadequate to maintain antitumor immunity. Therapeutic blockade of the T cell negative regulator programmed death-ligand 1 (PD-L1, also called B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 was upregulated in the tumor microenvironment after IR. Administration of anti-PD-L1 enhanced the efficacy of IR through a cytotoxic T cell-dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti-PD-L1 synergistically reduced the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which suppress T cells and alter the tumor immune microenvironment. Furthermore, activation of cytotoxic T cells with combination therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy.

Progression of Barrett's Metaplasia to Adenocarcinoma Is Associated with the Suppression of the Transcriptional Programs of Epidermal Differentiation

We did expressional profiling on 24 paired samples of normal esophageal epithelium, Barretts metaplasia, and esophageal adenocarcinomas. Matching tissue samples representing the three different histologic types were obtained from each patient undergoing esophagectomy for adenocarcinoma. Our analysis compared the molecular changes accompanying the transformation of normal squamous epithelium with Barretts esophagus and adenocarcinoma in individual patients rather than in a random cohort. We tested the hypothesis that expressional profiling may reveal gene sets that can be used as molecular markers of progression from normal esophageal epithelium to Barretts esophagus and adenocarcinoma. Expressional profiling was done using U133A GeneChip (Affymetrix), which represent approximately two thirds of the human genome. The final selection of 214 genes permitted the discrimination of differential gene expression of normal esophageal squamous epithelium, Barretts esophagus, and adenocarcinoma using two-dimensional hierarchical clustering of selected genes. These data indicate that transformation of Barretts esophagus to adenocarcinoma is associated with suppression of the genes involved in epidermal differentiation, including genes in 1q21 loci and corresponding to the epidermal differentiation complex. Correlation analysis of genes concordantly expressed in Barretts esophagus and adenocarcinoma revealed 21 genes that represent potential genetic markers of disease progression and pharmacologic targets for treatment intervention. PCR analysis of genes selected based on DNA array experiments revealed that estimation of the ratios of GATA6 to SPRR3 allows discrimination among normal esophageal epithelium, Barretts dysplasia, and adenocarcinoma.

MicroRNA Expression Characterizes Oligometastasis(es)

Background Cancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by ≤5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy. Methods Here, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy. Results Patients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression. Conclusions These results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment.

Signal Transducer and Activator of Transcription 1 Regulates Both Cytotoxic and Prosurvival Functions in Tumor Cells

Elsewhere, we reported that multiple serial in vivo passage of a squamous cell carcinoma cells (SCC61) concurrent with ionizing radiation (IR) treatment resulted in the selection of radioresistant tumor (nu61) that overexpresses the signal transducer and activator of transcription 1 (Stat1)/IFN-dependent pathway. Here, we report that (a) the Stat1 pathway is induced by IR, (b) constitutive overexpression of Stat1 is linked with failure to transmit a cytotoxic signal by radiation or IFNs, (c) selection of parental cell line SCC61 against IFN-alpha and IFN-gamma leads to the same IR- and IFN-resistant phenotype as was obtained by IR selection, and (d) suppression of Stat1 by short hairpin RNA renders the IR-resistant nu61 cells radiosensitive to IR. We propose a model that transient induction of Stat1 by IFN, IR, or other stress signals activates cytotoxic genes and cytotoxic response. Constitutive overexpression of Stat1 on the other hand leads to the suppression of the cytotoxic response and induces prosurvival genes that, at high levels of Stat1, render the cells resistant to IR or other inducers of cell death.

STAT1 Pathway Mediates Amplification of Metastatic Potential and Resistance to Therapy

Background Traditionally IFN/STAT1 signaling is connected with an anti-viral response and pro-apoptotic tumor-suppressor functions. Emerging functions of a constitutively activated IFN/STAT1 pathway suggest an association with an aggressive tumor phenotype. We hypothesized that tumor clones that constitutively overexpress this pathway are preferentially selected by the host microenvironment due to a resistance to STAT1-dependent cytotoxicity and demonstrate increased metastatic ability combined with increased resistance to genotoxic stress. Methodology/Principal Findings Here we report that clones of B16F1 tumors grown in the lungs of syngeneic C57BL/6 mice demonstrate variable transcriptional levels of IFN/STAT1 pathway expression. Tumor cells that constitutively overexpress the IFN/STAT1 pathway (STAT1H genotype) are selected by the lung microenvironment. STAT1H tumor cells also demonstrate resistance to IFN-gamma (IFNγ), ionizing radiation (IR), and doxorubicin relative to parental B16F1 and low expressors of the IFN/STAT1 pathway (STAT1L genotype). Stable knockdown of STAT1 reversed the aggressive phenotype and decreased both lung colonization and resistance to genotoxic stress. Conclusions Our results identify a pathway activated by tumor-stromal interactions thereby selecting for pro-metastatic and therapy-resistant tumor clones. New therapies targeted against the IFN/STAT1 signaling pathway may provide an effective strategy to treat or sensitize aggressive tumor clones to conventional cancer therapies and potentially prevent distant organ colonization.

Oligo- and Polymetastatic Progression in Lung Metastasis(es) Patients Is Associated with Specific MicroRNAs

Rationale Strategies to stage and treat cancer rely on a presumption of either localized or widespread metastatic disease. An intermediate state of metastasis termed oligometastasis(es) characterized by limited progression has been proposed. Oligometastases are amenable to treatment by surgical resection or radiotherapy. Methods We analyzed microRNA expression patterns from lung metastasis samples of patients with ≤5 initial metastases resected with curative intent. Results Patients were stratified into subgroups based on their rate of metastatic progression. We prioritized microRNAs between patients with the highest and lowest rates of recurrence. We designated these as high rate of progression (HRP) and low rate of progression (LRP); the latter group included patients with no recurrences. The prioritized microRNAs distinguished HRP from LRP and were associated with rate of metastatic progression and survival in an independent validation dataset. Conclusion Oligo- and poly- metastasis are distinct entities at the clinical and molecular level.

Radiation-induced equilibrium is a balance between tumor cell proliferation and T cell-mediated killing.

Local failures following radiation therapy are multifactorial, and the contributions of the tumor and the host are complex. Current models of tumor equilibrium suggest that a balance exists between cell birth and cell death due to insufficient angiogenesis, immune effects, or intrinsic cellular factors. We investigated whether host immune responses contribute to radiation-induced tumor equilibrium in animal models. We report an essential role for immune cells and their cytokines in suppressing tumor cell regrowth in two experimental animal model systems. Depletion of T cells or neutralization of IFN-γ reversed radiation-induced equilibrium, leading to tumor regrowth. We also demonstrate that PD-L1 blockade augments T cell responses, leading to rejection of tumors in radiation-induced equilibrium. We identify an active interplay between tumor cells and immune cells that occurs in radiation-induced tumor equilibrium and suggest a potential role for disruption of the PD-L1/PD-1 axis in increasing local tumor control.

Poly(ADP-Ribose) Polymerase Inhibitor Induces Accelerated Senescence in Irradiated Breast Cancer Cells and Tumors

Persistent DNA double-strand breaks (DSB) may determine the antitumor effects of ionizing radiation (IR) by inducing apoptosis, necrosis, mitotic catastrophe, or permanent growth arrest. IR induces rapid modification of megabase chromatin domains surrounding DSBs via poly-ADP-ribosylation, phosphorylation, acetylation, and protein assembly. The dynamics of these IR-induced foci (IRIF) have been implicated in DNA damage signaling and DNA repair. As an IRIF reporter, we tracked the relocalization of green fluorescent protein fused to a chromatin binding domain of the checkpoint adapter protein 53BP1 after IR of breast cancer cells and tumors. To block DSB repair in breast cancer cells and tumors, we targeted poly(ADP-ribose) polymerase (PARP) with ABT-888 (veliparib), one of several PARP inhibitors currently in clinical trials. PARP inhibition markedly enhanced IRIF persistence and increased breast cancer cell senescence both in vitro and in vivo, arguing for targeting IRIF resolution as a novel therapeutic strategy.

Glioblastoma cells block radiation-induced programmed cell death of endothelial cells.

We demonstrate that human umbilical vein endothelial cells (HUVEC) grown in co‐culture (CC) with U87 glioblastoma cells transfected with green fluorescent protein (GFP‐U87) exhibit resistance to radiation‐mediated apoptosis. cDNA macroarray analysis reveals increases in the accumulation of RNAs for HUVEC genes encoding cell adhesion molecules, growth factor‐related proteins, and cell cycle regulatory/DNA repair proteins. An increase in protein expression of integrin αv, integrin β1, MAPK(p42), Rad51, DNA‐PKCS, and ataxia telangiectasia gene (ATM) was detected in HUVEC grown in CC with GFP‐U87 cells compared with HUVEC grown in mono‐culture. Treatment with anti‐VEGF antibody decreases the expression of integrin αv, integrin β1, DNA‐PKCS and ATM with a corresponding increase in ionizing radiation (IR)‐induced apoptosis. These data support the concept that endothelial cells growing in the tumor microenvironment may develop resistance to cytotoxic therapies due to the up‐regulation by tumor cells of endothelial cells genes associated with survival.

Transcriptional control of viral gene therapy by cisplatin

Ionizing radiation (IR) and radical oxygen intermediates (ROIs) activate the early growth response-1 (Egr1) promoter through specific cis-acting sequences termed CArG elements. Ad.Egr.TNF.11D, a replication-deficient adenoviral vector containing CArG elements cloned upstream of the cDNA for human recombinant TNF-alpha was used to treat human esophageal adenocarcinoma and rat colon adenocarcinoma cells in culture and as xenografts in athymic nude mice. Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating ROIs. The present studies demonstrate induction of TNF-alpha production in tumor cells and xenografts treated with the combination of Ad.Egr.TNF.11D and cisplatin. The results show that the Egr1 promoter is induced by cisplatin and that this induction is mediated in part through the CArG elements. These studies also demonstrate an enhanced antitumor response without an increase in toxicity following treatment with Ad.Egr.TNF.11D and cisplatin, compared with either agent alone. Chemo-inducible cancer gene therapy thus provides a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents.