Background

Late-onset cardiomyopathy accounts for up to 50% of cancer unrelated deaths in cancer survivors. As cancer and anticancer therapy may impact on the heart we developed a reversible melanoma model in mice that allows to distinguish between cancer and therapy induced effects on the heart after recovery.

Methods

Male C57BL/6 mice (11 weeks of age) were injected with B16F10 melanoma cells that stably expressed herpes simplex virus thymidine kinase (HSVtk) and a luciferase reporter gene, thus enabling luciferin dependent visualization of tumor growth by in vivo imaging system (IVIS) and tumor cell specific apoptosis induction by ganciclovir (GCV). At advanced cancer stage (HSVtk-M, Tab.1) GCV treatment was started until tumor signal loss in IVIS (HSVtk+GCV-M, Tab.1). Healthy control mice received GCV (GCV-M, Tab.1). A subset received moderate doses of doxorubicin (DOX, 3 doses of 4mg/kg body weight (BW)) before GCV therapy (HSVtk+GCV+DOX-M, Tab.1). Follow up of surviving mice was 5±1 weeks after tumor signal loss. Cardiac function (echocardiography) and left ventricular (LV) gene expression (RNA-Seq. followed by DAVID pathway analyses) were compared. In neonatal rat cardiomyocytes (NRCM) treated with BMAL1 siRNA and DOX apoptosis markers (cleaved PARP and Caspase 3) were analyzed in western blots.

Results

Advanced cancer induced significant loss of BW, heart weight (HW), skeletal muscle weight (SKW) and cardiac function (fractional shortening (FS), Tab.1) compared to GCV-M. Cancer induced cardiomyopathy was associated with altered expression of 3724 genes (p-adj.<0.01) affecting 87 pathways in LV tissue. After GCV-induced tumor elimination HW, SKW, cardiac function (Tab.1) and gene expression pattern had recovered to GCV-M levels. Treatment of tumor-bearing mice with moderate dose of DOX before GCV treatment did not impair cardiac function acutely. However, at follow-up healed HSVtk+GCV+DOX-M displayed lower BW (Tab.1) and a trend to increased tumor-independent mortality (HSVtk+GCV+DOX-M: 33% vs. HSVtk+GVC-M 8%) compared to HSVtk+GCV-M. Surviving HSVtk+GCV+DOX-M displayed 123 differently expressed genes in LV tissue compared to HSVtk+GCV-M (p-adj.<0.05) with the circadian rhythm pathway (i.e. BMAL1 -65%, PER1/2 +100% vs HSVtk+GCV-M, p-adj.<0.01) emerging as the most altered pathway. In NRCM with siRNA-mediated BMAL1 reduction DOX treatment led to significantly more apoptosis (cl.PARP/PARP ratio +2-fold; cl.Casp.3/Casp.3 ratio +2.8-fold, p<0.01) compared to control-siRNA NRCM+DOX.

Conclusion

DOX-free tumor elimination resulted in nearly complete recovery from cancer induced morphologic, functional and molecular changes. In turn, tumor treatment with DOX induced long-term adverse effects with reduced BW and increased tumor independent mortality. Moreover, it caused long-lasting alterations in cardiac gene expression program with impairment in central circadian rhythm genes. This in turn promotes cardiomyocyte death in response to additional stressors such as DOX treatment.

*p<0.05, **p<0.01 vs GCV-M; #p<0.05, ##p<0.01 vs HSVtk-M