Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90
FRONTIERS IN ONCOLOGY
S100A10 is a critical mediator of GAS6/AXL-induced angiogenesis in renal cell carcinoma.
Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells.
2019; 8 (1): e1523097
Angiogenesis is a hallmark of cancer that promotes tumor progression and metastasis. However, antiangiogenic agents have limited efficacy in cancer therapy due to the development of resistance. In clear cell renal cell carcinoma (ccRCC), AXL expression is associated with antiangiogenic resistance and poor survival. Here, we establish a role for GAS6/AXL signaling in promoting the angiogenic potential of ccRCC cells through the regulation of the plasminogen receptor S100A10. Genetic and therapeutic inhibition of AXL signaling in ccRCC tumor xenografts reduced tumor vessel density and growth under the renal capsule. GAS6/AXL signaling activated the expression of S100A10 through SRC to promote plasmin production, endothelial cell invasion and angiogenesis. Importantly, treatment with the small molecule AXL inhibitor cabozantinib or an ultra-high affinity soluble AXL Fc fusion decoy receptor (sAXL) reduced the growth of a pazopanib-resistant ccRCC patient-derived xenograft. Moreover, the combination of sAXL synergized with pazopanib and axitinib to reduce ccRCC patient-derived xenograft growth and vessel density. These findings highlight a role for AXL/S100A10 signaling in mediating the angiogenic potential of ccRCC cells and support the combination of AXL inhibitors with antiangiogenic agents for advanced ccRCC.
View details for DOI 10.1158/0008-5472.CAN-19-1366
View details for PubMedID 31585940
A novel HSP90 inhibitor with reduced hepatotoxicity synergizes with radiotherapy to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer
2016; 7 (28): 43199-43219
The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as in situ cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20Gy, 4*2Gy, 2Gy, 0Gy) with cell lines of triple-negative breast cancer in vitro and in vivo. Previously, we have shown that especially high single doses (20Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes in vivo. Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses - at least in models of triple-negative breast cancer.
View details for PubMedID 30546963
Targeting the heat shock response in combination with radiotherapy: Sensitizing cancer cells to irradiation-induced cell death and heating up their immunogenicity
2015; 368 (2): 209-229
The chaperone heat shock protein 90 (HSP90) crucially supports the maturation, folding, and stability of a variety of client proteins which are of pivotal importance for the survival and proliferation of cancer cells. Consequently, targeting of HSP90 has emerged as an attractive strategy of anti-cancer therapy, and it appears to be particularly effective in the context of molecular sensitization towards radiotherapy as has been proven in preclinical models of different cancer entities. However, so far the clinical translation has largely been hampered by suboptimal pharmacological properties and serious hepatotoxicity of first- and second-generation HSP90 inhibitors. Here, we report on NW457, a novel radicicol-derived member of the pochoxime family with reduced hepatotoxicity, how it inhibits the DNA damage response and how it synergizes with ionizing irradiation to induce apoptosis, abrogate clonogenic survival, and improve tumor control in models of colorectal cancer in vitro and in vivo.
View details for DOI 10.18632/oncotarget.9774
View details for Web of Science ID 000385395700028
View details for PubMedID 27259245
HSP90 inhibition as a means of radiosensitizing resistant, aggressive soft tissue sarcomas
2015; 365 (2): 211-222
Radiotherapy represents an essential treatment option for the majority of cancer patients in different stages of their disease. Physical achievements of the recent years led to the implementation of high precision treatment planning procedures, and image-guided dose delivery is current state of the art. Yet, radiotherapy still faces several limitations with cancer intrinsic radioresistance being a key driver of therapeutic failure. Accordingly, the mechanisms orchestrating radioresistance and their therapeutic targeting by combined modality approaches are in the center of attention of numerous radiation oncologists. In the present review, we summarize and discuss therapeutic approaches that exploit the heat shock response, either by hyperthermia or by pharmacological heat shock protein inhibition, in combination with radiotherapy. These strategies appear particularly promising, since they sensitize cancer cells to irradiation-induced cell death and at the same time have proven the potential to promote systemic anti-tumor immune mechanisms, which may target not only locally surviving tumor cells, but also distant out-of-field metastases.
View details for DOI 10.1016/j.canlet.2015.02.047
View details for Web of Science ID 000361864200010
View details for PubMedID 25754814
Low and moderate doses of ionizing radiation up to 2 Gy modulate transmigration and chemotaxis of activated macrophages, provoke an anti-inflammatory cytokine milieu, but do not impact upon viability and phagocytic function
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
2015; 179 (1): 50-61
Radiotherapy is an essential part of multi-modal treatment for soft tissue sarcomas. Treatment failure is commonly attributed to radioresistance, but comprehensive analyses of radiosensitivity are not available, and suitable biomarkers or candidates for targeted radiosensitization are scarce. Here, we systematically analyzed the intrinsic radioresistance of a panel of soft tissue sarcoma cell lines, and extracted scores of radioresistance by principal component analysis (PCA). To identify molecular markers of radioresistance, transcriptomic profiling of DNA damage response regulators was performed. The expression levels of HSP90 and its clients ATR, ATM, and NBS1 revealed strong, positive correlations with the PCA-derived radioresistance scores. Their functional involvement was addressed by HSP90 inhibition, which preferentially sensitized radioresistant sarcoma cells and was accompanied by delayed ?-H2AX foci clearance and HSP90 client protein degradation. The induction of apoptosis and necrosis was not significantly enhanced, but increased levels of basal and irradiation-induced senescence upon HSP90 inhibition were detected. Finally, evaluation of our findings in the TCGA soft tissue sarcoma cohort revealed elevated expression levels of HSP90, ATR, ATM, and NBS1 in a relevant subset of cases with particularly poor prognosis, which might preferentially benefit from HSP90 inhibition in combination with radiotherapy in the future.
View details for DOI 10.1016/j.canlet.2015.05.024
View details for Web of Science ID 000357756100009
View details for PubMedID 26044951
Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated gamma-irradiation
Benign painful and inflammatory diseases have been treated for decades with low/moderate doses of ionizing radiation (LD-X-irradiation). Tissue macrophages regulate initiation and resolution of inflammation by the secretion of cytokines and by acting as professional phagocytes. Having these pivotal functions, we were interested in how activated macrophages are modulated by LD-X-irradiation, also with regard to radiation protection issues and carcinogenesis. We set up an ex-vivo model in which lipopolysaccharide pre-activated peritoneal macrophages (pM?) of radiosensitive BALB/c mice, mimicking activated macrophages under inflammatory conditions, were exposed to X-irradiation from 0ˇ01 Gy up to 2 Gy. Afterwards, the viability of the pM?, their transmigration and chemotaxis, the phagocytic behaviour, the secretion of inflammatory cytokines and underlying signalling pathways were determined. Exposure of pM? up to a single dose of 2 Gy did not influence their viability and phagocytic function, an important fact regarding radiation protection. However, significantly reduced migration, but increased chemotaxis of pM? after exposure to 0ˇ1 or 0ˇ5 Gy, was detected. Both might relate to the resolution of inflammation. Cytokine analyses revealed that, in particular, the moderate dose of 0ˇ5 Gy applied in low-dose radiotherapy for inflammatory diseases results in an anti-inflammatory cytokine microenvironment of pM?, as the secretion of the proinflammatory cytokine interleukin (IL)-1? was reduced and that of the anti-inflammatory cytokine transforming growth factor (TGF)-? increased. Further, the reduced secretion of IL-1? correlated with reduced nuclear translocation of nuclear factor (NF)-?B p65, starting at exposure of pM? to 0ˇ5 Gy of X-irradiation. We conclude that inflammation is modulated by LD-X-irradiation via changing the inflammatory phenotype of macrophages.
View details for DOI 10.1111/cei.12344
View details for Web of Science ID 000345603000008
View details for PubMedID 24730395
Current concepts in clinical radiation oncology
RADIATION AND ENVIRONMENTAL BIOPHYSICS
2014; 53 (1): 1-29
Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells.Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards ?-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry.Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to ?-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39.Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches.
View details for DOI 10.1186/1748-717X-9-85
View details for Web of Science ID 000335436300001
View details for PubMedID 24666643
Serum-Derived Plasminogen Is Activated by Apoptotic Cells and Promotes Their Phagocytic Clearance
JOURNAL OF IMMUNOLOGY
2012; 189 (12): 5722-5728
Based on its potent capacity to induce tumor cell death and to abrogate clonogenic survival, radiotherapy is a key part of multimodal cancer treatment approaches. Numerous clinical trials have documented the clear correlation between improved local control and increased overall survival. However, despite all progress, the efficacy of radiation-based treatment approaches is still limited by different technological, biological, and clinical constraints. In principle, the following major issues can be distinguished: (1) The intrinsic radiation resistance of several tumors is higher than that of the surrounding normal tissue, (2) the true patho-anatomical borders of tumors or areas at risk are not perfectly identifiable, (3) the treatment volume cannot be adjusted properly during a given treatment series, and (4) the individual heterogeneity in terms of tumor and normal tissue responses toward irradiation is immense. At present, research efforts in radiation oncology follow three major tracks, in order to address these limitations: (1) implementation of molecularly targeted agents and 'omics'-based screening and stratification procedures, (2) improvement of treatment planning, imaging, and accuracy of dose application, and (3) clinical implementation of other types of radiation, including protons and heavy ions. Several of these strategies have already revealed promising improvements with regard to clinical outcome. Nevertheless, many open questions remain with individualization of treatment approaches being a key problem. In the present review, the current status of radiation-based cancer treatment with particular focus on novel aspects and developments that will influence the field of radiation oncology in the near future is summarized and discussed.
View details for DOI 10.1007/s00411-013-0497-2
View details for Web of Science ID 000331977300001
View details for PubMedID 24141602
Dying cell clearance and its impact on the outcome of tumor radiotherapy.
Frontiers in oncology
2012; 2: 116-?
The elimination of apoptotic cells, called efferocytosis, is fundamentally important for tissue homeostasis and prevents the onset of inflammation and autoimmunity. Serum proteins are known to assist in this complex process. In the current study, we performed a multistep chromatographic fractionation of human serum and identified plasminogen, a protein involved in fibrinolysis, wound healing, and tissue remodeling, as a novel serum-derived factor promoting apoptotic cell removal. Even at levels significantly lower than its serum concentration, purified plasminogen strongly enhanced apoptotic prey cell internalization by macrophages. Plasminogen acted mainly on prey cells, whereas on macrophages no enhancement of the engulfment process was observed. We further demonstrate that the efferocytosis-promoting activity essentially required the proteolytic activation of plasminogen and was completely abrogated by the urokinase plasminogen activator inhibitor-1 and serine protease inhibitor aprotinin. Thus, our study assigns a new function to plasminogen and plasmin in apoptotic cell clearance.
View details for DOI 10.4049/jimmunol.1200922
View details for Web of Science ID 000311995800028
View details for PubMedID 23150713
The induction of tumor cell death is one of the major goals of radiotherapy and has been considered to be the central determinant of its therapeutic outcome for a long time. However, accumulating evidence suggests that the success of radiotherapy does not only derive from direct cytotoxic effects on the tumor cells alone, but instead might also depend - at least in part - on innate as well as adaptive immune responses, which can particularly target tumor cells that survive local irradiation. The clearance of dying tumor cells by phagocytic cells of the innate immune system represents a crucial step in this scenario. Dendritic cells and macrophages, which engulf, process and present dying tumor cell material to adaptive immune cells, can trigger, skew, or inhibit adaptive immune responses, respectively. In this review we summarize the current knowledge of different forms of cell death induced by ionizing radiation, the multi-step process of dying cell clearance, and its immunological consequences with special regard toward the potential exploitation of these mechanisms for the improvement of tumor radiotherapy.
View details for DOI 10.3389/fonc.2012.00116
View details for PubMedID 22973558