Regulatory role of sphingolipid metabolites, sphingosine-1-phosphate and C16:0 ceramide, in immunogenic cell death of colon cancer cells induced by Bak/Bax-activation

ABSTRACT We have previously identified sphingolipid metabolism as a key intracellular process associated with immunogenic cell death (ICD) induced by mitoxantrone in colon cancer cells. Specifically, we have demonstrated that inhibition of the sphingosine kinases (SphKs) synergistically enhanced production of hallmarks of ICD including ectoCRT production. To better understand the mechanism associated with ICD enhanced by SphK1-inhibition, we focused on the ER stress-associated intracellular signaling pathways leading to ectoCRT production. It is known that ABT-263 and AZD-5991 (ABT/AZD) are inhibitors of Bcl-2/Bcl-XL and MCL-1, respectively, leading to activation of Bak/Bax. Herein, we now provide evidence that treatment of DLD-1 colon cancer cells with ABT/AZD results in the production of ectoCRT indicative of ICD. Additionally, our data show that ABT/AZD-induced ectoCRT production is significantly enhanced by combination treatment with the SphK1 inhibitor, PF-543. Mechanistically, we demonstrate that combined treatment of ABT/AZD+PF-543 induces ectoCRT exposure in a caspase 8-dependent manner. Accordingly, we have identified a Bak/Bax activation-dependent pathway that leads to activation of a pro-survival SphK1/sphingosine-1-phosphate (S1P) signaling that attenuates ectoCRT production. Additionally, we have identified a regulatory role of ceramide synthase 6 (CerS6)- C16:0 Cer in transporting of dimeric CRT to the cell surface (ectoCRT). Together, these results indicate that sphingolipid metabolites, such as S1P and C16:0 Cer, have a key regulatory role for survival/death decisions of cancer cells in response to ICD-inducing chemotherapeutic agents such as mitoxantrone and ABT/AZD. Hence, targeting SphKs may be an innovative means to enhance the efficacy of ICD-inducing chemotherapeutic agents promoting anti-tumor innate/adaptive immune response since SphK inhibition blocks the anti-ICD effects of S1P while simultaneously accumulating sphingosine (Sph) leading to pro-ICD C16:0 Cer synthesis.