Myoferlin silencing affects mitochondria function in cells derived from adult T-cell leukemia patients

Abstract

Human T-cell leukemia virus (HTLV-1) is a retrovirus that infects mainly CD4+ T-cells. Infection with HTLV-1 can lead to either adult T-cell leukemia, a fatal cancer, or a form of HTLV-associated myelopathy, a disease that affects the host nervous system. Once integrated, the virus expresses several viral proteins. Among them, HBZ has been shown to be important for the maintenance of HTLV-1 infected cells. HBZ can regulate the transcription of several cellular genes, and we previously reported that HBZ strongly upregulates the expression of a cellular gene known as myoferlin. Myoferlin is mainly expressed in muscle cells but has also been reported to be abnormally expressed in pancreatic and triple-negative breast cancers. In these cells, myoferlin plays a role in endosomal trafficking and cellular metabolism. Additionally, the role of myoferlin in maintaining mitochondria functions has recently been reported in pancreatic cancer cells. In HTLV-1 infected T-cells, we previously reported that myoferlin increases HTLV-1 infectivity and invasiveness of these cells. Another aspect of our research is to understand whether myoferlin also plays a role in the metabolism of HTLV-1 infected T-cells and, more specifically, is important for mitochondria function. We used three different cell lines, one that was established in vitro (SLB-1), and two derived from ATL patients (ATL-2 and TL-Om1). We knocked-down myoferlin using a small hairpin RNA (shMYOF) and used shGFP to establish control cell lines. Using these cell lines, we found that myoferlin was purified with mitochondria fractions, and its localization was reduced when myoferlin was silenced. We analyzed mitochondria presence in shGFP and shMYOF cell lines by labeling the mitochondria with a TOM20 antibody, that recognizes the TOM complex found on the outer membrane of the mitochondria; or by using MitoTracker Deep Red FM, a compound that is an indicator of the membrane potential of the mitochondria. We found that labeling with TOM20 antibody was similar between all cell lines. However, staining with MitoTracker Deep Red FM was significantly reduced in myoferlin depleted cells. These results suggest that HTLV-1 infected cells expressing myoferlin have an impaired membrane potential. Surprisingly, when we assessed ATP production from these cells, we only found a decrease in shMYOF from the ATL-derived cells, ATL-2 and TL-Om1, while the in vitro established cells, SLB-1, showed an increase in ATP production. We correlated the decrease in MitoTracker Deep Red FM staining in SLB-1 shMYOF to an increase in expression of the drugs efflux pump ABCB1/MDR1. The MitoTracker Deep Red FM staining was restored when we use a specific inhibitor of this pump in the SLB-1 shMYOF cells. It is intriguing that SLB-1 and ATL-derived cells exhibit different levels of ATP production when myoferlin is silenced, but the reduction in ATP production observed in ATL-derived cells is promising and suggest that myoferlin could be targeted to affect the metabolism of these cells.