Ocular Melanoma and Treatment with Metformin
Metformin Activity
Metformin is one of the most common medications for patients with type 2 diabetes mellitus. It is an antihyperglycemic agent that activates the adenosine monophosphate-activated kinase (AMPK). Through this, a person’s cellular ATP levels are restored. The activation of AMPK by metformin suggests that the improvement in intracellular metabolic profiles could be related to autophagic induction.
Metformin upregulates autophagic activity through direct phosphorylation of UNC51-like kinase 1 and Beclin1, which are key factors involved in the initiation of autophagy. In addition, the nicotinamide adenine dinucleotide-dependent deacetylase Sirtuin 1 (SIRT1) was found to be significantly upregulated after AMPK phosphorylation, inducing autophagy upon glucose starvation.
Although most studies have shown that metformin activates autophagic flux, metformin can also downregulate autophagy and alleviate hyperglycemia-induced endothelial impairment in a Hedgehog pathway-dependent manner. In addition, metformin sensitizes lung cancer cells by inhibiting autophagy. Thus, metformin plays a dual role in autophagy induction, but further investigations are required to determine their relationships.
Recent studies have found that metformin may also be used to reduce cancer risk for patients with diabetes. Repurposing metformin as cancer treatment is currently being done to a wide range of clinical trials for different kinds of cancer. Recent analyses have found that metformin reduces the proliferation of cancer cells. It’s also being used effectively with radiotherapy to reduce tumor growth in a variety of animal models.
Ocular Melanoma and Metformin Treatment
Ocular melanoma is a life-threatening kind of malignancy found in one’s eye. It’s also the most common primary malignancy of the eye in adults. Ocular melanoma is resistant to the most common types of chemotherapy and radiotherapy making it one of the hardest to treat. When metastasis occurs, the average overall survival is less than 1 year. Although metformin has been shown to induce tumor-specific inhibition in different cancers, the role of metformin in ocular melanoma remains unclear.
This study explores the utility of exploiting the tumor suppressing activity of metformin to treat ocular melanoma via its mechanism of action involving autophagy regulation.
Results
Metformin induces tumor-specific inhibition in ocular melanomas both in-vitro and in vivo.
To test the efficacy of different concentrations of metformin in controlled pigmented cells and ocular melanoma cells, the researchers first analyzed the IC50 of metformin in these cells. The IC50 in ocular melanoma cells (MUM2B, MEL290, CRMM1, and CM2005.1) was approximately 1000μM; however, the IC50 in normal pigmented cells was over 2600μM.
Moreover, we found that 1000μM (1mM) metformin did not influence the proliferation of normal control cells, while that of most ocular melanoma cells was significantly inhibited. In addition, metformin-treated ocular melanoma cells formed smaller and fewer colonies, while normal control cells remained unaffected.
To verify the role of metformin-induced ocular melanoma inhibition in vivo, the researchers then established an orthotopic UM xenograft model with a luciferase tag. Animal imaging showed that metformin-treated MUM2B cells presented with a significantly decreased intensity of bioluminescence.
Metformin attenuates autophagic flux in ocular melanoma cells.
As metformin plays an important part in autophagy regulation, the researchers tested autophagy flux after metformin treatment in ocular melanoma cells. Unexpectedly, metformin-treated melanoma cells presented decreased levels of LC3II/I ratio, Beclin1 and ATG5, and increased p62 in a dose-dependent and time-dependent manner, which indicated that metformin acted as an autophagy inhibitor in ocular melanoma cells.
OPTN is a candidate target of metformin for autophagy inhibition.
Through a high-throughput proteomic assay of 3106 proteins with 19 352 identified peptides, we identified 47 upregulated proteins and 53 downregulated proteins. These differentially expressed proteins were mainly found in mitochondrial and ribosomal-associated proteins.
Among these proteins, OPTN was the only protein that has been proven to be an activator of autophagy. The researchers then chose OPTN as the candidate target of metformin that could be responsible for the inhibition of autophagy.
OPTN promoted tumourigenesis of ocular melanoma in vitro and in vivo
As OPTN was significantly upregulated in tumors and modulated autophagic flux in ocular melanoma, the researchers explored the role of OPTN in the tumor progression of ocular melanoma. Notably, the OPTN-silenced group formed fewer and smaller colonies than the scramble RNA group.
Additionally, the CCK-8 assay showed an attenuated proliferation rate after OPTN inhibition in MUM2B and CRMM1 cell lines. Moreover, the migration ability was significantly attenuated in OPTN-silenced tumor cells.
Furthermore, we verified the function of OPTN in ocular melanoma in vivo through an intraocular xenograft tumor model combined with luciferase animal imaging. The results showed that OPTN-inhibited MUM2B cells presented with a significantly decreased bioluminescence intensity. Taken together, these results showed that OPTN was an oncogene in ocular melanoma.
Reintroduction of OPTN partially rescued metformin-induced autophagic inhibition
To further verify the relationship between metformin-guided autophagy inhibition and OPTN expression, the researchers rescued OPTN expression after metformin treatment in ocular melanoma cells by overexpressing OPTN. Through RT-qPCR analysis, the researchers found that OPTN expression returned to the basal level after metformin treatment under simultaneous treatment with the OPTN overexpression vector.
More importantly, after reintroducing OPTN expression, the researchers found that the decreased autophagy levels were restored, along with an elevatedLC3 II/I ratio, Beclin1, and ATG5 levels and decreased p62level compared to those in metformin-treated cells.
Metformin epigenetically silenced OPTN through histone deacetylation in the OPTN promoter
OPTN was significantly downregulated after treatment with metformin, the researchers explored the mechanism underlying metformin-induced OPTN down-regulation. Because histone acetylation is an epigenetic modification involved in the maintenance of gene expression, the researchers hypothesized that OPTN could be downregulated by the loss of his tone acetylation.
Conclusion
The study reveals that metformin is a promising agent for the treatment of ocular melanomas. The working concentration of 1 mM would be clinically feasible through intravitreal drug delivery, which allows reaching high drug concentrations in the vitreous cavity and avoiding adverse effects of systemic drug administration.
Metformin also inhibited the tumorigenesis of ocular melanoma by suppressing autophagic flux. Autophagy is a highly conserved catabolic process involving the formation of autophagosomes that engulf cellular organelles and proteins passing to the lysosome. Because autophagy plays a dual function in cancer, the modulation of autophagy during tumourigenesis, either by reversing cytoprotective autophagy or by promoting cytotoxic autophagy, could potentially overcome cancer resistance to common chemotherapy.
In conclusion, metformin significantly inhibited the tumor progression of ocular melanoma. Metformin also acts as an autophagy inhibitor through histone deacetylation OPTN, which serves as a novel oncogene in ocular melanoma. These studies provide novel insights into metformin-guided tumor suppression of malignant ocular melanoma and the potential mechanism underlying the dual role of metformin in autophagy regulation.
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