Role of ROS in sustaining cell survival signaling in classical Hodgkin's lymphoma

Abstract

Hodgkin’s lymphoma is one of the most frequent lymphomas in the western world, frequently occurring in relatively young patients with a median age of diagnosis at 38. The disease is characterized by the presence of mononucleated Hodgkin's cells and multinucleated Reed-Sternberg cells. Classical Hodgkin's lymphoma (cHL) derives survival signaling and evades immune surveillance by recruitment of various hematopoietic cells to the tumor microenvironment by secretion of various chemokines and cytokines. This highly reactive microenvironment results in an increased generation of reactive oxygen species (ROS) in cHL and therefore, the biological role of ROS in cHL was investigated using cHL cell lines. To identify cell intrinsic mechanism of ROS generation in cHL, the expression levels of the ROS regulatory genes such as NOX1, NOX3, NOX5, SOD1, SOD2 and GPX1 were analyzed. The expression levels of SOD1 and SOD2 were significantly higher in cHL cell lines compared to normal B cells (LCLs) and DLBCL cell lines while the expression of GPX1 which is involved in ROS scavenging was significantly lower in cHL. To evaluate if the presence of ROS was important for cHL cell survival, the cHL cell lines were treated with antioxidants and NOX inhibitors (iNOX) which resulted in cell death by apoptosis as shown by annexin V/PI FACS and caspase 3 cleavage. Induction of cell death upon treatment with antioxidants and iNOX was found to be mediated by the blockage of the constitutively active JAK/STAT signaling, which is one of the key survival signaling pathways in cHL. Also, inhibition of ROS generation was found to augment NF-κB in cHL cell lines, indicating a crucial role of ROS in the regulation of JAK/STAT and NF-κB pathways. To further prove that antioxidants and iNOX treatment specifically targets JAK/STAT signaling, cells were transfected with constitutively active STAT6VT mutant, followed by antioxidant and iNOX treatment. Reinforced JAK/STAT activation was able to partly rescue cell death induced by reduction in ROS generation. To further characterize the mechanism leading to the attenuation of JAK/STAT signaling upon ROS inhibition, the negative regulators of JAK/STAT signaling were analyzed. One of the frequently inactivated negative regulators of JAK/STAT is PTP1B and therefore the redox regulation of PTP1B was verified. Analysis of PTP1B activity indicated that cHL cell lines have very low PTP1B activity compared to that of control LCL B cell lines and primary lymphocytes. Using NEM and DTT, the amount of oxidized (inactive) PTP1B in cHL cell lines was analyzed and all cHL cell lines except U-HO1 were found to be associated with high levels of PTP1B in the oxidized form, rendering them inactive. Therefore it was verified if the loss of PTP1B would impair cHL response to ROS reduction. Knockdown of PTPN1/PTP1B using siRNA led to a partial rescue of cell death following treatment with antioxidants and iNOX. Interestingly, also the knockdown of PTPN2/TC-PTP was found to have a similar effect as that of PTPN1 loss, indicating that ROS could oxidatively inactivate various protein tyrosine phosphatases in cHL. The augmentation of NF-κB signaling by the inhibition of ROS generation was reflected by changes in transcriptional levels of various cytokines relevant in cHL. Taken together, ROS in cHL mediates various key cell survival pathways chiefly by oxidative inactivation of the negative regulators of the signaling. Reduction in ROS also resulted in variations in NF-κB signaling and global changes in cytokine profiles. The results indicate an important pro-survival function of ROS which could potentially be exploited for treatment of cHL.