Abstract 4618: TGF-B upregulates GSK3B and mediates NK dysfunction in cancer
Authors: Ojo Evelyn , Otegbeye Folashade , Moreton Stephen , Wald David 
Published: July 2017

Transforming growth factor-B (TGF-B) is an important regulator of tumorigenesis. Initially, it was discovered to have anti-cancer activities by promoting apoptosis and preventing hyperproliferation of cells. However, abundant presence of TGF-B in the tumor microenvironment is known to hinder cancer eradication by suppressing anti-tumor activities of immune cells. The molecular switches that regulate the role of TGF-B are not fully understood. One main target of TGF-B-mediated immune cell dysfunction are natural killer (NK) cells. NK cells are innate immune cells that lyse virally-infected and cancer cells. Recently, we have discovered that glycogen synthase kinase 3-beta (GSK3B) upregulation in NK cells leads to NK dysfunction. Another study has indicated that GSK3 phosphorylates SMAD3 to inhibit TGF-B-mediated growth dysfunction in AML12 cells. In addition, GSK3B inhibition has been linked to increased sensitivity of cancer cells to NK killing but the role of GSK3B in NK function is understudied. This study examines how the role that GSK3B has on TGF-B-mediated NK cell dysfunction. Methods: NK cells were isolated from the peripheral blood of healthy donors and expanded in vitro for 14 days. NK cells were either treated with or without 5ng/mL TGF-B for 72 hours, and GSK3B expression, and the expression of NK activating receptors was measured via flow cytometry. In addition, GSK3B expression is abrogated using a siRNA in NK cells. The NK cells were treated with or without TGF-B, and NK cell cytotoxicity is measured with a fluorometric cytotoxicity assay. Results: TGF-B pretreatment of NK cells led to 2-fold GSK3B expression in NK cells. TGF-B pretreatment led to decreased expression of NK activating receptors NKp46 and NKG2D. Also, TGF-B led to decreased expression of lymphocyte function-associated antigen-1 (LFA-1), an adhesion molecule necessary for NK cells to adhere to target cells prior to lysis of the target cells. TGF-B pretreatment of NK cells led to 33.3% reduced killing of both leukemic and solid tumor targets. Pharmacologic and genetic abrogation of GSK3 minimized TGF-B-mediated NK dysfunction as measured by calcein-AM based cytotoxicity assays. Importantly, inhibiting GSK3B activity with TGF-B treatment rescued NK cell cytotoxic function. Discussion: Our data suggests that TGF-B leads to upregulated GSK3B expression, and downregulation of NK activating receptors. In addition, TGF-B reduced NK killing of cancer cells. We believe that TGF-B-mediated NK dysfunction is facilitated by GSK3B upregulation. Further studies are underway to determine how TGF-B affects the expression of other NK receptors, and to elucidate the mechanism by which TGF-B directs GSK3B upregulation.

Citation Format: Evelyn Ojo, Folashade Otegbeye, Stephen Moreton, David Wald. TGF-B upregulates GSK3B and mediates NK dysfunction in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2017-4618