Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated gamma-irradiation

Background: 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. Methods: 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. Results: 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. Conclusions: 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

The Retinitis Pigmentosa Mutation c.3444+1G>A in CNGB1 Results in Skipping of Exon 32

Retinitis pigmentosa (RP) is a severe hereditary eye disorder characterized by progressive degeneration of photoreceptors and subsequent loss of vision. Two of the RP associated mutations were found in the CNGB1 gene that encodes the B subunit of the rod cyclic nucleotide-gated channel (CNGB1a). One of them (c.3444+1G>A) is located at the donor site of exon 32 and has been proposed to result in a frameshift and truncation of the last 28 aa of the corresponding protein. However, this ambiguous conclusion was not verified by experimental data. Recently, another study reported that the last 28 aa of CNGB1a harbor a motif required for the proper targeting of this subunit to rod photoreceptor outer segments. This suggests that defective targeting is the major cause for the RP phenotype in affected patients. Here, we investigated the splicing of c.3444+1G>A by exon trapping experiments and could demonstrate that instead of the proposed truncation of the last 28 aa this mutation leads to replacement of the last 170 aa of CNGB1a by 68 unrelated amino acids. The 170 aa deletion covers the complete distal C-terminus including the last 10 aa of an important alpha (αC) helix within the ligand-binding domain of CNGB1a. When expressed in a heterologous expression system the corresponding mutant full-length CNGB1a subunit was more susceptible to proteosomal degradation compared to the wild-type counterpart. In conclusion, our experimental data do not support the hypothesis proposed by the original study on the c.3444+1G>A mutation. Based on this, we suggest that apart from the defective targeting other mechanisms may be responsible for the RP phenotype in affected individuals

Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms – a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition – apart from its radiosensitizing potential – obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC

HSP90 inhibition as a means of radiosensitizing resistant, aggressive soft tissue sarcomas

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 γ-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

c.3444+1G>A mutation affects the splicing and expression of CNGB1.

<p>(A) Schematic representation of the minigene construct used for the exon trapping experiment showing the position of the c.3444+1G>A mutation (marked by an arrowhead) and the deleted intronic <i>Xba</i>I-fragment. Vector backbone sequence is depicted in green. (B) Revese transcriptase PCR from HEK293T cells transfected with mutant and wild type minigene constructs. The electropherogram for the c.3444+1G>A mutant shows the skipping of exon 32. (C) Scheme showing the splice products. The length of the respective PCR products is indicated by double arrows. (D) Schematic comparison of the WT and mutant protein demonstrating the lack of the entire distal C-terminus and the last 10 aa of the αC helix in the context of the c.3444G>A mutation. Skipping of exon 32 causes a frameshift which results in addition of 68 unrelated amino acids after aa position 1075 of the CNGB1a protein (highlighted in grey). The numbers represent the length of the respective proteins (1245 aa for WT and 1143 for the mutant). (E) Western blot of membranes isolated from HEK293T cells transfected with CNGA1 and wild type or mutant CNGB1a probed with anti-B1 (<i>top panel</i>) or anti-ATPase (<i>bottom panel</i>). The weaker expression of the mutant protein was normalized in the presence of the proteasome inhibitors MG-132 and ALLN. CNBD: cyclic nucleotide-binding domain. Primers are shown as arrows. S1–S6: transmembrane segments; WT: wild type, Mut: c.3444+1G>A mutation.</p

Priming anti-tumor immunity by radiotherapy: Dying tumor cell-derived DAMPs trigger endothelial cell activation and recruitment of myeloid cells

The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as 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 (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer and . Previously, we have shown that especially high single doses (20 Gy) 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 . 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 20 Gy-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

uPA‐PAI‐1 heteromerization promotes breast cancer progression by attracting tumorigenic neutrophils

Abstract High intratumoral levels of urokinase‐type plasminogen activator (uPA)‐plasminogen activator inhibitor‐1 (PAI‐1) heteromers predict impaired survival and treatment response in early breast cancer. The pathogenetic role of this protein complex remains obscure. Here, we demonstrate that heteromerization of uPA and PAI‐1 multiplies the potential of the single proteins to attract pro‐tumorigenic neutrophils. To this end, tumor‐released uPA‐PAI‐1 utilizes very low‐density lipoprotein receptor and mitogen‐activated protein kinases to initiate a pro‐inflammatory program in perivascular macrophages. This enforces neutrophil trafficking to cancerous lesions and skews these immune cells toward a pro‐tumorigenic phenotype, thus supporting tumor growth and metastasis. Blockade of uPA‐PAI‐1 heteromerization by a novel small‐molecule inhibitor interfered with these events and effectively prevented tumor progression. Our findings identify a therapeutically targetable, hitherto unknown interplay between hemostasis and innate immunity that drives breast cancer progression. As a personalized immunotherapeutic strategy, blockade of uPA‐PAI‐1 heteromerization might be particularly beneficial for patients with highly aggressive uPA‐PAI‐1high tumors