Date Published: April 10, 2019
Publisher: Public Library of Science
Author(s): Su Wu, Anders M. Näär, Joseph T. Nickels.
While investigating the role played by de novo lipid (DNL) biosynthesis in cancer cells, we sought a medium condition that would support cell proliferation without providing any serum lipids. Here we report that a defined serum free cell culture medium condition containing insulin, transferrin and selenium (ITS) supports controlled study of transcriptional regulation of de novo fatty acid (DNFA) production and de novo cholesterol synthesis (DNCS) in melanoma cell lines. This lipid-free ITS medium is able to support continuous proliferation of several melanoma cell lines that utilize DNL to support their lipid requirements. We show that the ITS medium stimulates gene transcription in support of both DNFA and DNCS, specifically mediated by SREBP1/2 in melanoma cells. We further found that the ITS medium promoted SREBP1 nuclear localization and occupancy on DNFA gene promoters. Our data show clear utility of this serum and lipid-free medium for melanoma cancer cell culture and lipid-related areas of investigation.
De Novo lipid (DNL) synthesis is the metabolic pathway that converts carbohydrates into fatty acids, cholesterol, phospholipids, triglycerides, and other cellular lipids required for normal cellular homeostasis and proliferation/growth. In healthy adults, DNL is for the most part restricted to liver and adipose tissues for energy storage or distribution to other tissues. Many malignant cancer cells also exhibit elevated DNL as a hallmark adaptation to support proliferation and survival [1, 2]. Among the DNL pathways, de novo fatty acid (DNFA) biosynthesis is of particular interest as a potential therapeutic target for cancers [3, 4]. DNFA is primarily regulated at the mRNA level of catalytic enzymes that drive the biosynthetic reactions , a transcriptional process under the control of the sterol regulatory element-binding protein 1 (SREBP1) . However, DNFA gene regulation is still not fully understood at the molecular level. Cell culture studies of DNFA with widely-accepted serum-containing medium conditions are often confounded by the presence of external lipids, with consequent difficulty to disentangle the respective effects of lipid synthesis and lipid update, both of which may occur even among cells that are able to survive and proliferate using DNFA alone [6, 7].
We report here that a serum-free and insulin-supplemented culture medium condition, 1% ITS supplemented RPMI-1640, supports DNFA and DNCS pathway activation as well as proliferation and survival of human melanoma cell lines. Under this condition, HT-144 cells proliferate while relying entirely on de novo lipid synthesis to meet lipid requirements. Expression of DNFA and DNCS enzymes increases significantly in cells when cultured in 1% ITS as compared with other cell culture conditions. We found that 1% ITS medium activates DNFA and DNCS gene expression through the transcription regulators SREBP1 and SREBP2, respectively. In particular, culturing cells in 1% ITS medium promoted the transcription activation of SREBP1 and accumulation of nuclear SREBP1 protein, as compared with other medium conditions, and cells cultured in 1% ITS medium exhibited further increased binding of SREBP1 at a DNFA gene promoter, consistent with high SREBP1-dependent gene activation under this medium condition.
In summary, we have identified and validated a serum-free and insulin supplemented (ITS) medium condition that is well suited for controlled study of lipogenic gene activation and its mechanism of action in melanomas, and perhaps other cancer cell types.