Glioma Cell Proliferation Promoted by AK4 Regulated mTORC1 Pathway Activation

A hallmark of cancer cell biology is the dysregulation of signaling pathways that manage cancer‐associated phenotypes such as proliferation and survival. One dysfunctional pathway in cancer cells is the mechanistic target of rapamycin (mTOR). The mTOR pathway, involving both mTOR Complex 1 (mTORC1) and mTORC2, functions as a central regulator of cell metabolism, growth, proliferation, and survival. mTORC1 kinase signaling allows for transitions between metabolic states by responding to environmental signals and controlling processes that regulate energy. Recently, the expression of an Adenylate Kinase 4 (AK4) protein has been identified as modulating cellular bioenergetics through the activation of pathways such as mTORC1, resulting in both enhanced proliferation and activation of stress responses. Additionally, reduced AK4 expression has been observed in primary human glioma cells, while the mTORC1 signaling pathway is highly activated in identical primary glioma cells. We hypothesize that AK4 expression regulates mTORC1 signaling and induces downstream effects that promote glioma cell biology. To determine if AK4 expression affects mTORC1 signaling, we knocked down AK4 expression using siRNA in U87‐MG glioma cell lines. Phospho‐specific modifications of activators and inhibitors upstream of the mTORC1 pathway and its downstream targets were assessed using Western blot (WB). AK4 knockdown (KD) and WB analysis revealed over‐active mTORC1 phosphorylation and inactivation of the downstream translational repressor, 4E‐BP1, compared to control glioma cell lines. Likewise, WB analysis displayed increased mTORC1 phosphorylation and activation of the downstream protein ribosomal S6, a regulator of protein translation. Reduced phosphorylation of upstream inhibitors and regulatory proteins of mTORC1, Tuberous Sclerosis Complex 2 (TSC2) and Regulatory‐associated protein of mTOR (RPTOR), was observed when compared to controls. Additionally, reduced mTORC1 phosphorylation of the autophagy inducing serine/threonine protein kinase ULK1 was evident by WB. Furthermore, bioluminescence analysis of AK4 KD U87‐MG glioma cells shows an increase in cell proliferation under nutrient stressed conditions compared to control. Overall, these data suggest that AK4 reduction may contribute to over‐active mTORC1 kinase signaling in glioma cells. Studies characterizing the role of AK4 reduction on glioma cell proliferation could further explain the contribution of endogenously reduced AK4 expression in glioma cell biology. By identifying the regulatory role of AK4, we hope to elucidate the therapeutic potential of modulating AK4 levels in cancer cells. Support or Funding Information NSF# HRD‐1463889.