Development of an exosomal gene signature to detect residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning.

Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups ("healthy", "osteosarcoma", "other bone tumor", or "non-neoplastic disease"). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as "osteosarcoma detected" or "osteosarcoma-NOT detected". Dogs in a validation set whose post-treatment samples were classified as "osteosarcoma-NOT detected" had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.

The beta adrenergic receptor antagonist propranolol alters mitogenic and apoptotic signaling in late stage breast cancer.

BACKGROUND: Substantial evidence supports the use of inexpensive ß-AR antagonists (beta blockers) against a variety of cancers, and the ß-AR antagonist propranolol was recently approved by the European Medicines Agency for the treatment of soft tissue sarcomas. Prospective and retrospective data published by our group and others suggest that non-selective ß-AR antagonists are effective at reducing proliferative rates in breast cancers, however the mechanism by which this occurs is largely unknown. METHODS: In this study, we measured changes in tumor proliferation and apoptosis in a late stage breast cancer patient treated with neoadjuvant propranolol. We expounded upon these clinical findings by employing an in vitro breast cancer model, where we used cell-based assays to evaluate propranolol-mediated molecular alterations related to cell proliferation and apoptosis. RESULTS: Neoadjuvant propranolol decreased expression of the pro-proliferative Ki-67 and pro-survival Bcl-2 markers, and increased pro-apoptotic p53 expression in a patient with stage III breast cancer. Molecular analysis revealed that ß-AR antagonism disrupted cell cycle progression and steady state levels of cyclins. Furthermore, propranolol treatment of breast cancer cells increased p53 levels, enhanced caspase cleavage, and induced apoptosis. CONCLUSION: Collectively, these data provide support for the incorporation of ß-AR antagonists into the clinical management of breast cancer, and elucidate a partial molecular mechanism explaining the efficacy of ß-AR antagonists against this disease.

Non-selective beta blockers inhibit angiosarcoma cell viability and increase progression free- and overall-survival in patients diagnosed with metastatic angiosarcoma.

Patients with metastatic angiosarcoma undergoing chemotherapy, radiation, and/or surgery experience a median progression free survival of less than 6 months and a median overall survival of less than 12 months. Given the aggressive nature of this cancer, angiosarcoma clinical responses to chemotherapy or targeted therapeutics are generally very poor. Inhibition of beta adrenergic receptor (ß-AR) signaling has recently been shown to decrease angiosarcoma tumor cell viability, abrogate tumor growth in mouse models, and decrease proliferation rates in preclinical and clinical settings. In the current study we used cell and animal tumor models to show that ß-AR antagonism abrogates mitogenic signaling and reduces angiosarcoma tumor cell viability, and these molecular alterations translated into patient tumors. We demonstrated that non-selective ß-AR antagonists are superior to selective ß-AR antagonists at inhibiting angiosarcoma cell viability. A prospective analysis of non- selective ß-AR antagonists in a single arm clinical study of metastatic angiosarcoma patients revealed that incorporation of either propranolol or carvedilol into patients' treatment regimens leads to a median progression free and overall survival of 9 and 36 months, respectively. These data suggest that incorporation of non-selective ß-AR antagonists into existing therapies against metastatic angiosarcoma can enhance clinical outcomes.

Use of non-selective ß-blockers is associated with decreased tumor proliferative indices in early stage breast cancer.

Previous studies suggest beta-adrenergic receptor (ß-AR) antagonists (ß-blockers) decrease breast cancer progression, tumor metastasis, and patient mortality; however the mechanism for this is unknown. Immunohistochemical analysis of normal and malignant breast tissue revealed overexpression of ß1-AR and ß3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of ß-blocker usage on tumor proliferation. Our analysis revealed that non-selective ß-blockers, but not selective ß-blockers, reduced tumor proliferation by 66% (p < 0.0001) in early stage breast cancer compared to non-users. We tested the efficacy of propranolol on an early stage breast cancer patient, and quantified the tumor proliferative index before and after treatment, revealing a propranolol-mediated 23% reduction (p = 0.02) in Ki67 positive tumor cells over a three-week period. The anti-proliferative effects of ß-blockers were measured in a panel of breast cancer lines, demonstrating that mammary epithelial cells were resistant to propranolol, and that most breast cancer cell lines displayed dose dependent viability decreases following treatment. Selective ß-blockers alone or in combination were not as effective as propranolol at reducing breast cancer cell proliferation. Molecular analysis revealed that propranolol treatment of the SK-BR-3 breast cancer line, which showed high sensitivity to beta blockade, led to a reduction in Ki67 protein expression, decreased phosphorylation of the mitogenic signaling regulators p44/42 MAPK, p38 MAPK, JNK, and CREB, increased phosphorylation of the cell survival/apoptosis regulators AKT, p53, and GSK3ß. In conclusion, use of non-selective ß-blockers in patients with early stage breast cancer may lead to decreased tumor proliferation.