14P - Lentivirally overexpressed c-Myc promoter binding protein (MBP-1) localizes in the cytoplasm of human cutaneous melanoma cell lines increasing cell proliferation and glycolysis rate

Background

C-Myc promoter binding protein (MBP-1) is a product of alternatively translated mRNA encoding alpha-enolase (ENO-1). In contrast to ENO-1, MBP-1 possesses no enzymatic activity but instead is able to bind P2 region of the c-Myc promoter leading to the modulation of its expression. Ectopic overexpression of MBP-1 was shown to reduce cell proliferation and tumorigenicity of numerous tumor cell lines, hence constituting an attractive target for cancer therapy.

Methods

We created lentiviral particles encoding HA-tagged, human MBP-1 protein, its C-terminal deletion mutant (MBP-1ΔC), or control, empty pRLL puro vector. With these tools, we created six stable transfectants derived from A375 and WM9 human melanoma cell lines. Detection of HA-tag by Western blot and immunofluorescence confirmed the overexpression of transfected proteins. We then used qPCR to estimate the effects of MBP-1 overexpression on the c-Myc transcription, Click-it Edu proliferation assay to assess the rate of cell proliferation, lactate detection assay in hypoxia and normoxia to measure the glycolytic rate and in vitro wound-healing assay to evaluate the migration ability of transduced cells.

Results

In our study, we found that overexpressed MBP-1 and MBP-1ΔC predominantly localized in the cytoplasm and only minimally decreased c-Myc mRNA expression, secondly, the proliferation rate of MBP-1- transduced cells increased in comparison to empty vector controls, and thirdly, the rate of glucose metabolism in normoxia and hypoxia increased in MBP-1 and MBP-1ΔC transduced cells. When assessing cell migration, we also found that overexpression of MBP-1 but not MBP-1ΔC led to a substantial decrease in the cell migration capacity of WM9 but not A375.

Conclusions

Our data underline potential pitfalls to avoid when overexpressing MBP-1 by means of lentiviral vectors. We provide evidence suggesting that lentiviral transduction of melanoma cell lines per se strongly affects cell proliferation and glucose metabolism. On the other hand, our research depicted an unexpected tumor-promoting activity of MBP-1 that can be largely dissociated from its nuclear localization and enzymatic activity.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Wroclaw Medical University, Wroclaw University of Environmental and Life Sciences.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.