Mutations of BRCA2 in canine mammary tumors and their targeting potential in clinical therapy.

Dogs develop cancer spontaneously with age, with breed-specific risk underlying differences in genetics. Mammary tumors are reported as the most frequent neoplasia in intact female dogs. Their high prevalence in certain breeds suggests a genetic component, as it is the case in human familial breast cancer, distinctly in BRCA2-associated cancers. However, the molecular genetics of BRCA2 in the pathogenesis of canine cancer are still under investigation.Genetic variations of canine BRCA2 comprised single nucleotide polymorphisms, insertions and deletions. The BRCA2 level has been shown to be reduced in tumor gland samples, suggesting that low expression of BRCA2 is contributing to mammary tumor development in dogs. Additionally, specific variations of the BRCA2 gene affect RAD51 binding strength, critically damage the BRCA2-RAD51 binding and further provoke a defective repair. In humans, preclinical and clinical data revealed a synthetic lethality interaction between BRCA2 mutations and PARP inhibition. PARP inhibitors are successfully used to increase chemo- and radiotherapy sensitivity, although they are also associated with numerous side effects and acquired resistance. Cancer treatment of canine patients could benefit from increased chemo- and radiosensitivity, as their cancer therapy protocols usually include only low doses of drugs or radiation. Early investigations show tolerability of iniparib in dogs. PARP inhibitors also imply higher therapy costs and consequently are less likely to be accepted by pet owners.We summarized the current evidence of canine BRCA2 gene alterations and their association with mammary tumors. Mutations in the canine BRCA2 gene have the potential to be exploited in clinical therapy through the usage of PARP inhibitors. However, further investigations are needed before introducing PARP inhibitors in veterinary clinical practice.

Role of HSP70 in response to (thermo)radiotherapy: analysis of gene expression in canine osteosarcoma cells by RNA-seq.

Pre-treatment of tumors with hyperthermia is often used to increase the efficacy of radiotherapy. One of the main proteins induced in response to hyperthermia is heat shock protein 70 (HSP70). The aim of our study was to investigate up- and down-regulated genes in response to (thermo)radiotherapy in HSP70 proficient and deficient canine osteosarcoma cell line (Abrams), and functional role of HSP70 in the mechanism of thermoradiosensitization. Cells were transfected with negative control siRNA or siRNA targeting HSP70 and treated with hyperthermia (HT), radiotherapy (RT), and thermoradiotherapy (HTRT). RNA sequencing was used to analyze gene expression. Hyperthermia and thermoradiotherapy, but not radiotherapy alone, induced differential gene expression. We identified genes differentially expressed only in HSP70 knockdown (thus HSP70-dependent) cells in response to hyperthermia and thermoradiotherapy. Interestingly, cell proliferation but not clonogenicity and apoptosis/necrosis was affected by the HSP70 knockdown in response to thermoradiotherapy. The results suggest that HSP70 regulates expression of specific genes in response to hyperthermia and thermoradiotherapy. Further investigations into the role of specific genes regulated in a HSP70-dependent manner in response to thermoradiotherapy could pave a way into new, combinatorial treatment options for (canine) osteosarcoma and other cancer types.

Methadone does not potentiate the effect of doxorubicin in canine tumour cell lines.

Opioid receptor activation was shown to enhance the efficacy of anti-neoplastic drugs in several human cancer cell lines. In these cell lines, doxorubicin increased the number of opioid receptors and methadone concurrently enhanced cellular doxorubicin uptake. Triggered through lay press and media, animal owners started to challenge veterinary oncologists with questions about methadone use in anti-cancer therapy. Especially in veterinary medicine, where side effects of chemotherapy are tolerated to a lesser extent and hence smaller doses are given, agents potentiating chemotherapeutic agents would be an optimal approach to treatment. Canine transitional cell carcinoma cells (TCC, K9TCC), canine osteosarcoma cells (OSA, Abrams) and canine hemangiosarcoma cells (HSA, DAL-4) were incubated with different combinations of methadone, buprenorphine and doxorubicin, in order to test inhibition of cell proliferation. Opioid receptor density was assessed with fluorescence-activated cell sorting in drug native and doxorubicin pretreated cells. In TCC and OSA cell lines opioid receptor density increased after doxorubicin pretreatment. In combination treatment, however, we did not find significant potentiation of doxorubicin's inhibitory effect on proliferation in these cell lines. Neither was there a significant increase of the effect of doxorubicin when the opioids were added 24 hr before doxorubicin. Hence, we could not confirm the hypothesis that opioids increase the anti-proliferative effect of the anti-neoplastic drug doxorubicin in any of these canine tumour cell lines. The lack of effect on a cellular level does not warrant a clinical approach to use opioids together with doxorubicin in dogs with cancer.

Holistic View on Cell Survival and DNA Damage: How Model-Based Data Analysis Supports Exploration of Dynamics in Biological Systems.

In this work, a method is established to calibrate a model that describes the basic dynamics of DNA damage and repair. The model can be used to extend planning for radiotherapy and hyperthermia in order to include the biological effects. In contrast to "syntactic" models (e.g., describing molecular kinetics), the model used here describes radiobiological semantics, resulting in a more powerful model but also in a far more challenging calibration. Model calibration is attempted from clonogenic assay data (doses of 0-6 Gy) and from time-resolved comet assay data obtained within 6 h after irradiation with 6 Gy. It is demonstrated that either of those two sources of information alone is insufficient for successful model calibration, and that both sources of information combined in a holistic approach are necessary to find viable model parameters. Approximate Bayesian computation (ABC) with simulated annealing is used for parameter search, revealing two aspects that are beneficial to resolving the calibration problem: (1) assessing posterior parameter distributions instead of point-estimates and (2) combining calibration runs from different assays by joining posterior distributions instead of running a single calibration run with a combined, computationally very expensive objective function.

Cell line-specific efficacy of thermoradiotherapy in human and canine cancer cells in vitro.

OBJECTIVE: Aims were to investigate sensitivity of various human and canine cancer cell lines to hyperthermia and the influence of particular treatment conditions, and to analyze the DNA-damage response and mode of cell death in cell line radiosensitized by hyperthermia. Additionally, we were interested in the involvement of HSP70 in radiosensitization. METHODS: Radiosensitization by hyperthermia was determined in a panel of human and canine cancer cell lines using clonogenic cell survival assay, as well as levels of heat shock proteins (HSPs) using immunoblotting. The influence of the hyperthermia-radiotherapy time gap, different temperatures and the order of treatments on clonogenicity of hyperthermia-sensitive A549 cells was investigated. Additionally, DNA damage and cell death were assessed by Comet assay and an apoptosis/necrosis assay. Further we induced transient knockdown in A549 cells to test HSP70's involvement in radiosensitization. RESULTS: Out of eight cell lines tested, only two (A549 and Abrams) showed significant decrease in clonogenic cell survival when pre-treated with hyperthermia at 42°C. Strong induction of HSP70 upon thermoradiotherapy (HT-RT) treatment was found in all cell lines. Transient knockdown of HSP70 in A549 cells did not result in decrease of clonogenic cell survival in response to HT-RT. CONCLUSION: Tumor cell-type, temperature and order of treatment play an important role in radiosensitization by hyperthermia. However, hyperthermia has limited potency to radiosensitize canine cancer cells grown in a 2D cell culture setting presented here. DNA damage and apoptosis/necrosis did not increase upon combined treatment and cytosolic levels of HSP70 appear not to play critical role in the radiosensitization of A549 cells.