Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.

The capacity of nicotine to affect the behavior of non-neuronal cells through neuronal nicotinic acetylcholine receptors (nAChRs) has been the subject of considerable recent attention. Previously, we showed that exposure to nicotine activates the nuclear factor of activated T cells (NFAT) transcription factor in lymphocytes and endothelial cells, leading to alterations in cellular growth and vascular endothelial growth factor production. Here, we extend these studies to document effects of nicotine on lymphocyte survival. The data show that nicotine induces paradoxical effects that might alternatively enforce survival or trigger apoptosis, suggesting that depending on timing and context, nicotine might act both as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. In addition, our results show that, while having overlapping functions, low and high affinity nAChRs also transmit signals that promote distinct outcomes in lymphocytes. The sum of our data suggests that selective modulation of nAChRs might be useful to regulate lymphocyte activation and survival in health and disease.

Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol enhanced doxorubicin cytotoxicity via antagonism of β-ARs or by preventing the lysosomal accumulation of doxorubicin. β-AR-like immunoreactivities were confirmed in primary tumor tissues and cell lines; receptor function was verified by monitoring downstream signaling pathways of β-ARs in response to receptor agonists and antagonists. Mechanistically, propranolol increased cytoplasmic doxorubicin concentrations in sarcoma cells by decreasing the lysosomal accumulation and cellular efflux of this chemotherapeutic agent. Equivalent concentrations of the receptor-active S-(−) and -inactive R-(+) enantiomers of propranolol produced similar effects, supporting a β-AR-independent mechanism. Long-term exposure of hemangiosarcoma cells to propranolol expanded both lysosomal size and number, yet cells remained sensitive to doxorubicin in the presence of propranolol. In contrast, removal of propranolol increased cellular resistance to doxorubicin, underscoring lysosomal doxorubicin sequestration as a key mechanism of resistance. Our results support the repurposing of the R-(+) enantiomer of propranolol with weak base chemotherapeutics to increase cytotoxicity and reduce the development of drug-resistant cell populations without the cardiovascular and other side effects associated with antagonism of β-ARs.

A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumor growth of feline oral squamous cell carcinoma

Introduction: We and others have found that propranolol, a beta adrenergic receptor antagonist used to treat heart disease, improved the overall survival of patients with angiosarcoma. While combination treatment with chemotherapies has further improved patient responses, identification of chemotherapy drugs that synergize with propranolol may refine treatment protocols. We previously showed that doxorubicin accumulates within the lysosomes of angiosarcoma cell lines, leading to drug resistance. Because propranolol is also lysosomotropic, we sought to determine whether propranolol could reduce the lysosomal accumulation of doxorubicin, increasing tumor cell chemosensitivity. Methods: We used angiosarcoma cell lines to evaluate the effects of doxorubicin in combination with propranolol on cell viability, doxorubicin retention, and drug efflux. The effects of the adrenergic agonist, epinephrine, on doxorubicin retention and efflux were also evaluated. Transmission electron microscopy and cell viability assays were used to assess the long-term effects of propranolol treatment on the lysosome system and doxorubicin resistance, respectively. Results: While propranolol sensitized cells to doxorubicin by reducing the lysosomal accumulation of the drug, it increased the cytosolic concentration of doxorubicin by inhibiting drug efflux. Epinephrine had the opposite effect, reducing the cytosolic accumulation of doxorubicin and activating drug efflux. Although long-term exposure to propranolol expanded both the size and number of cellular lysosomes, cells remained sensitive to doxorubicin treatment in the presence of propranolol; however, removal of propranolol increased drug resistance above previous levels. Conclusions: Angiosarcoma cells sequestered doxorubicin in cellular lysosomes and enhanced drug efflux to circumvent cytotoxicity. Propranolol countered these processes by altering the intracellular distribution of doxorubicin, allowing higher concentrations of drug to reach its nuclear target. Removal of propranolol allowed doxorubicin to re-accumulate in lysosomes, increasing drug resistance. While our results support the use of lysosomotropic chemotherapy drugs in combination with propranolol to increase cytotoxic effects, they also suggest concurrent administration with chemotherapy to reduce the development of resistant cell populations. These results may aid in the rational design of other lysosomotropic chemotherapies in combination with propranolol to treat angiosarcomas. Citation Format: Jhuma Saha, Jong-Hyuk Kim, Clarissa Amaya, Caleb Witcher, Derek M. Korpela, Ali Khammanivong, Josephine Taylor, Brad A. Bryan, Erin B. Dickerson. Propranolol alters the intracellular accumulation of doxorubicin and sensitizes angiosarcoma cells to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-013.