ABSTRACT
Sphingolipid and ganglioside metabolic pathways are crucial components of cell signalling, having established roles in tumor cell proliferation, invasion, and migration. However, regulatory mechanisms controlling sphingolipid and ganglioside biosynthesis in mammalian cells is less known. Here, we show that RICTOR, the regulatory subunit of mTORC2, regulates the synthesis of sphingolipids and gangliosides in luminal breast cancer-specific MCF-7 and BT-474 cells through transcriptional and epigenetic mechanisms. RICTOR regulates glucosylceramide levels by modulating the expression of UDP-Glucose Ceramide Glucosyl transferase (UGCG). We identify Zinc Finger protein X-linked (ZFX) as a RICTOR-responsive transcription factor whose recruitment to the UGCG promoter is regulated by DNA methyltransferases and histone demethylase (KDM5A) that are known AKT substrates. We further demonstrate that RICTOR regulates the synthesis of GD3 gangliosides through ZFX and UGCG, and triggers the activation of EGFR signalling pathway, thereby promoting tumor growth. In line with our findings in cell culture and mice models, we observe an elevated expression of RICTOR, ZFX, and UGCG in Indian luminal breast cancer tissues, and in TCGA and METABRIC datasets. Together, we establish a key regulatory circuit, RICTOR-AKT-ZFX-UGCG-Ganglioside-EGFR-AKT, and elucidate its contribution to breast cancer progression.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We validated our studies in another breast cancer cell line (BT-474 cell line) that represents luminal B breast cancer subtype, and revised the manuscript extensively. Now, our results clearly demonstrate the molecular mechanism and signalling circuitry of mTOR/RICTOR regulating ganglioside metabolism in two luminal breast cancer cell lines (MCF-7 and BT-474) supported by murine data. To further validate if ZFX-mediated UGCG regulation is a common phenomenon among other cancer cell types, we showed that ZFX silencing downregulates UGCG expression in other cancer cell types like HCT-116 (colon cancer), HepG2 (liver cancer), and MDA-MB-453 (HER2+ breast cancer representative). This has added the dimension of robustness to our already validated metabolic-signalling-gene regulatory circuit in breast cancer subtypes. In addition, we also performed the validation/rescue studies using gangliosides. In the revised manuscript, we show that addition of GD3 gangliosides on MCF-7_RICTORSH and BT-474_RICTORSH cells enhances the cell proliferation, and addition of GM1 gangliosides on MCF-7_UGCGOE, MCF-7_ZFXOE, BT-474_UGCGOE, and BT-474_ZFXOE cells decreases cell proliferation. This provides more confidence to the existing data that has established the circuit connecting mTORC2/RICTOR to ganglioside metabolism controlling tumor progression. We have included these results in the revised manuscript.
