Down-regulation Of OLA1,a Major Regulator Of Integrated Stress Response, Potentiates Tumor Growth And Metastasis

Tumor cells are located in a dynamic microenvironment. Self-sufficiency in growth signals, ability to evading growth suppressors and cell death as well as sustained angiogensis is necessary to make cancers become malignant, invasive and metastatic. In consideration of fast growth of tumors, massive stresses from either the extracellular or intracellular sources are produced. Outgrowing the blood supply will deprived tumors of nutrients and oxygen. Although tumors cells have tolerance to nutrient deprivation, energy metabolism is inhibited and parts of cells die. Hypoxia could cause necrosis and apoptosis inside cells together with DNA degradation. When protein homeostasis in the endoplasmic reticulum (ER) lumen is perturbed, unfolded protein response (UPR) induced by ER stress decreases protein synthesis and causes growth arrest. Usually carcinoma cells are under persistent oxidative stress. Excessive reactive oxygen species (ROS) production can lead to cell death. To survive under a large number of unfavorable factors, cancer cells have developed a superior stress response system referred as integrated stress response (ISR). Eukaryotic translation initiation factor 2a (eIF2a) is known to play important roles in ISR. Phosphorylation of eIF2a leads to shutdown of global protein synthesis but enhances translation of some special stress proteins such as activating transcription factor 4(ATF4). Increased level of ATF4 would trigger a gene expression program referred as ISR via transcriptional regulation. Activation of GCN2-eIF2a-ATF4 leaded to the elevated expression of asparagine synthetase (ASNS) which enhanced the uptake of nutrient. The prolonged activation of transcription factor C/EBP Homology Protein (CHOP) would cause stress damage through repression of BCL2 and the induction of proapoptotic genes.ISR is an important process that helps cancer cells to overcome the stresses. Meanwhile, sustained activation of ISR could cause cell death through inhibition of protein translation and activation of apoptosis.OLA1, Obg-like ATPase 1, belongs to the YchF subfamily of Obg-like GTPases. OLA1 is predicted to be a regulatory protein that interacts with downstream effector to function in the process of protein translation and degradation. Neverless, its function and related mechanism are poorly understood. In this study, we sought to identify OLAl’s role as a novel regulator of ISR in tumor progression through in vivo and in vitro study.Stable 231-D3H2-LN cell line was constructed through transduction of lentiviral OLAl-shRNA particles. Equal amount of stable 231-D3H2-LN-Control/OLA1-knockdown cells were injected to fat pad of nude mouse. Three weeks after inoculation, OLA 1-depleted tumors significantly grew faster than Control group. OLA1-deficient tumors were bigger and heavier. Interestingly,OLA1 has minor effect on growth of cancer cell line in vitro. Another inoculation of stable 231-D3H2-LN was done to fat pad of the SCID mice.On the last day, lymph node metastases were detected by IVIS (In Vivo Imaging Systems) in 84.6%(11/13) of OLA 1-depleted group compared to 38.5% (5/13) of Control. Subsequent histopathology was applied to confirm metastases in lymph nodes and lungs. There were more lymph nodes (20 to 9) and lung metastasis (6 to 2) in OLAl-defecient tumors. Knockdown of OLAl enhanced tumor metastasis. Together, all the in vivo study of breast cancer xenograft models point to a significant and novel role of OLA 1 in regulating the tumor’s progression.To confirm OLAl’s function in regulation the growth and metastasis of tumors, we screened a panel of proteins and found deregulation of p-GSK-3β,β-p70S6K, p-eIF2a, ASNS and CHOP in OLA 1-deficient tumor tissues. IHC staining was further deployed. Decreased expression of CHOP and reduced apoptosis (TUNNEL) were seen in OLA1-depleted tumors. Ki67 staining result revealed that there was no correlation between OLA1 expression and proliferation. All these findings convinced us that the pathway in ISR changes within tumor tissues.We hypothesized that OLA1 functions as a regulator of IRS. In in-vitro study, we treated cancer cell lines with different stress inducers. Notably, under serum starvation, amino acid starvation, ER stress (tunicamycin) or oxidative stress (H2O2), the survival of OLA 1-depleted 231-D3H2-LN cells was much better than Control. Down-regulation of OLA1 increased breast cancer cell lines’resistance to these four stresses. Western blot results revealed that lower level of p-eIF2a and p-GSK-3β was associated with decreased OLA1 expression when treated with 72h serum starvation. In response to full amino acid deprivation, decreased activation of GCN2-p-eIF2α-ATF4 was found in the OLA1-depleted 231-D3H2-LN. Consistently, under ER stress or oxidative stress, reduced activation of PERK- p-eIF2α-ATF4-ANSN was noticed in 231-D3H2-LN with OLA1-depletion.35S labeling kit was used to measure the global protein translation rate. Under serum starvation or ER stress,OLA1-depleted 231-D3H2-LN presented higher 35S incorporation compared to Control. These in vitro findings corroborated previous observations in vivo and suggested attenuation of ISR and enhanced capability of survive under stress after down-regulation of OLA 1 in breast cancer cell lines.A rescue experiment was devised in OLA 1-deficient cells. Stable HeLa and 231-D3H2-LN with depletion of OLA1 were transfected with Flag-only or FLAG-OLA-res. Importantly, reconstituted OLA1 expression in OLA 1-deficient cells elevated PERK-eIF2a-ATF4 expression in ISR pathway. To further explore the mechanism how down-regulation of OLA 1 decreased the expression of key proteins in ISR pathway, OLA1-wild type, OLA1-N230A and OLA1-△TGS into the pCMV-Tagl vector with an N-terminal FLAG tag were designed. HEK-293T cells were transient transfected with these 3 plasimds. Immunoprecipitation followed by western blot analysis strongly suggest that OLA1 directly interacts with eIF2a.To address whether the expression of OLA1 has any correlation with clinical features of breast cancer, the optimized immunohistochemistry was applied.160 cases of patients who underwent surgery and chemotherapy were analyzed. In Kaplan-Meier analysis, lower expression of OLA1 was associated with higher chance of cancer relapse and poor survive (log-rank:P= 0.024 and 0.033). The multivariate Cox proportional hazard analysis revealed that low expression of OLA1 indicated more cancer relapse in patients with breast cancer (HR:0.540; 95% CI:0.345-0.844; P=0.007) and poor survive (HR:0.514; 95% CI:0.292-0.905; P=0.021). Lower expression of OLA 1 predicted poor prognosis in patients with breast cancer.Our results establish that OLA1 regulates the process of ISR and may serve as a potential prognostic biomarker for breast cancer.