Urszula Kazimierczak

Phosphorylation of the translation initiation factor eIF2α at serine 51 determines the cell fate decisions of Akt in response to oxidative stress

Phosphorylation of the α subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP) is a master regulator of cell adaptation to various forms of stress with implications in antitumor treatments with chemotherapeutic drugs. Herein, we demonstrate that genetic loss of the eIF2α kinases PERK and GCN2 or impaired eIF2αP by genetic means renders immortalized mouse fibroblasts as well as human tumor cells increasingly susceptible to death by oxidative stress. We also show that eIF2αP facilitates Akt activation in cells subjected to oxidative insults. However, whereas Akt activation has a pro-survival role in eIF2αP-proficient cells, the lesser amount of activated Akt in eIF2αP-deficient cells promotes death. At the molecular level, we demonstrate that eIF2αP acts through an ATF4-independent mechanism to control Akt activity via the regulation of mTORC1. Specifically, eIF2αP downregulates mTORC1 activity, which in turn relieves the feedback inhibition of PI3K resulting in the upregulation of the mTORC2-Akt arm. Inhibition of mTORC1 by rapamycin restores Akt activity in eIF2αP-deficient cells but renders them highly susceptible to Akt-mediated death by oxidative stress. Our data demonstrate that eIF2αP acts as a molecular switch that dictates either cell survival or death by activated Akt in response to oxidative stress. Hence, we propose that inactivation of eIF2αP may be a suitable approach to unleash the killing power of Akt in tumor cells treated with pro-oxidant drugs.

Captopril, an Angiotensin-Converting Enzyme Inhibitor, Promotes Growth of Immunogenic Tumors in Mice

Purpose: Antitumor potential of angiotensin-converting enzyme inhibitors has been shown in different preclinical settings, which always involved immunocompromised organisms or nonimmunogenic tumor models. In our study, we wanted to evaluate the effect of captopril on growth of immunogenic tumors in immunocompetent animals. Experimental Design: We used different murine tumor models to evaluate the effect of captopril on tumor take and survival of tumor-bearing immunocompetent and immunocompromised mice. We used an orthotopic renal cell cancer model and highly immunogenic tumor model, which were based on kidney subcapsular injection of RenCa cells or s.c. injection of MethA cells, respectively. To show the influence of captopril on antigen-specific immune responses, we have used two model antigens (green fluorescent protein and β-galactosidase). Results: Captopril decreased survival of RenCa-bearing, immunocompetent mice in a dose-dependent manner and in adjuvant setting. In nephrectomized mice, captopril shortened their survival. Captopril promoted formation of immunogenic MethA sarcoma tumors but had no effect on nonimmunogenic melanoma cells (B78-H1). Treatment of immunocompromised mice bearing MethA tumors or RenCa kidney tumors with captopril did not affect tumor formation nor survival, respectively. Captopril-treated mice immunized with AdLacZ or AdGFP vectors did not generate or generated decreased numbers of antigen-specific CD8+ T cells, respectively. However, they showed B-cell responses represented by infiltration of MethA tumors with activated B cells and dramatically increased serum level of β-galactosidase-specific antibodies. Conclusions: Our results show a novel role of captopril in tumor biology and the tumor-promoting properties of captopril seem to be associated with its immunomodulatory potential.

mTORC2 Balances AKT Activation and eIF2α Serine 51 Phosphorylation to Promote Survival under Stress

The mTOR nucleates two complexes, namely mTOR complex 1 and 2 (mTORC1 and mTORC2), which are implicated in cell growth, survival, metabolism, and cancer. Phosphorylation of the α-subunit of translation initiation factor eIF2 at serine 51 (eIF2αS51P) is a key event of mRNA translation initiation and a master regulator of cell fate during cellular stress. Recent studies have implicated mTOR signaling in the stress response, but its connection to eIF2αS51P has remained unclear. Herein, we report that genetic as well as catalytic inhibition of mTORC2 induces eIF2αS51P. On the other hand, the allosteric inhibitor rapamycin induces eIF2αS51P through pathways that are independent of mTORC1 inactivation. Increased eIF2αS51P by impaired mTORC2 depends on the inactivation of AKT, which primes the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK. The biologic function of eIF2αS51P was characterized in tuberous sclerosis complex (TSC)-mutant cells, which are defective in mTORC2 and AKT activity. TSC-mutant cells exhibit increased PERK activity, which is downregulated by the reconstitution of the cells with an activated form of AKT1. Also, TSC-mutant cells are increasingly susceptible to ER stress, which is reversed by AKT1 reconstitution. The susceptibility of TSC-mutant cells to ER stress is further enhanced by the pharmacologic inhibition of PERK or genetic inactivation of eIF2αS51P. Thus, the PERK/eIF2αS51P arm is an important compensatory prosurvival mechanism, which substitutes for the loss of AKT under ER stress. Implications: A novel mechanistic link between mTOR function and protein synthesis is identified in TSC-null tumor cells under stress and reveals potential for the development of antitumor treatments with stress-inducing chemotherapeutics. Mol Cancer Res; 13(10); 1377–88. ©2015 AACR.

The eIF2α serine 51 phosphorylation-ATF4 arm promotes HIPPO signaling and cell death under oxidative stress

The HIPPO pathway is an evolutionary conserved regulator of organ size that controls both cell proliferation and death. This pathway has an important role in mediating cell death in response to oxidative stress through the inactivation of Yes-associated protein (YAP) and inhibition of anti-oxidant gene expression. Cells exposed to oxidative stress induce the phosphorylation of the alpha (α) subunit of the translation initiation factor eIF2 at serine 51 (eIF2αP), a modification that leads to the general inhibition of mRNA translation initiation. Under these conditions, increased eIF2αP facilitates the mRNA translation of activating transcription factor 4 (ATF4), which mediates either cell survival and adaptation or cell death under conditions of severe stress. Herein, we demonstrate a functional connection between the HIPPO and eIF2αP-ATF4 pathways under oxidative stress. We demonstrate that ATF4 promotes the stabilization of the large tumor suppressor 1 (LATS1), which inactivates YAP by phosphorylation. ATF4 inhibits the expression of NEDD4.2 and WWP1 mRNAs under pro-oxidant conditions, which encode ubiquitin ligases mediating the proteasomal degradation of LATS1. Increased LATS1 stability is required for the induction of cell death under oxidative stress. Our data reveal a previously unidentified ATF4-dependent pathway in the induction of cell death under oxidative stress via the activation of LATS1 and HIPPO pathway.

Hyper-interleukin-11 novel designer molecular adjuvant targeting gp130 for whole cell cancer vaccines

Background: Hyper-IL-11 (H11) is a fusion protein comprising IL-11 and soluble IL-11 receptor directly targeting gp130. We evaluated efficacy of H11 as a molecular adjuvant in therapeutic whole tumor cell vaccine formulation. Methods: H11 was tested in ectopic and orthotopic murine renal cell carcinoma (RENCA) models. H11 cDNA was transduced into RENCA cells (RENCA-H11). Mice were immunized with RENCA-H11 or control vaccine (RENCA-IRR) in prophylactic, adjuvant and therapeutic settings. Tumor formation, survival and immune mechanisms activated by H11 were studied. Results: Biologically active H11 was secreted by RENCA-H11 cells. Immunization with RENCA-H11 resulted in mounting specific anti-RENCA response. Treatment of tumor bearing mice in adjuvant setting prevented disease recurrence in therapeutic setting eradicated tumors. In induction phase H11 inhibited T-regulatory cell formation and activated recruitment and maturation of dendritic cells. Downstream of immunization tumors were densely infiltrated by CD8+, CD4+, NK cells, cells expressing CD8+CD69+ and CD4+CD62Llow. Conclusions: H11 is a good candidate for adjuvant of whole tumor cell vaccines. Direct targeting of gp130 leads to induction of specific and long lasting anticancer immune response. Enhancement of tumor antigen presentation, abrogation of immune tolerance, and activation of NK cells and generation of memory cells lead to eradication of existing tumors.

Cell-based Hyper-interleukin 6 or Hyper-interleukin 11 secreting vaccines combined with low dose cyclophosphamide in an orthotopic murine prostate cancer model.

Background Cell based vaccines encoding Hyper-IL-6 (H6) and Hyper-IL-11 (H11) present high activity in murine melanoma and renal cancer model. We evaluated the efficacy of cellular vaccines modified with H6 or H11 combined with cyclophosphamide in orthotopic murine prostate cancer model. Material and methods TRAMP cells were transduced with H6 and H11 cDNA (TRAMP-H6 and TRAMP-H11). An orthotopic TRAMP model based on the implantation of TRAMP cells into the dorsolateral lobe of the prostate of C57BL6/J mice was employed. The efficacy of TRAMP-H6 and TRAMP-H11 vaccines evaluated in the therapeutic setting was compared with the TRAMP cells modified with a mock transduced E1-deleted adenoviral vector (TRAMP-AdV) and non-modified irradiated TRAMP cells (TRAMP IRR) in relation to naive (non-immunized) mice. In the next experimental groups mice vaccinated with TRAMP-H6 and TRAMP-H11 received cyclophosphamide (CY). Detection of immune cells in the spleen in mice receiving vaccines combined with CY was evaluated. Results Modification of TRAMP cells with H6 increased the efficacy of TRAMP-based whole-cell vaccine. The highest response rate was observed in mice receiving TRAMP-H6 alone and combined with CY. Vaccination with TRAMP-H6 alone and combined with CY and TRAMP H11 combined with CY extended median OS of mice bearing orthotopic TRAMP tumors in therapeutic setting. Low dose CY administered alone demonstrated some antitumor activity in employed model. TRAMP-H6 or TRAMP-H11 combined with CY strongly augmented generation of CD8+, CD4+ T lymphocytes and memory T cells. Immunization with TRAMP combined with or without CY suppressed generation of T regulatory cells. Conslusions Prostate cancer vaccines modified with H6 or H11 induce prostate tumour regression and increase mice survival by stimulating the immune system. Cyclophosphamide added to modified TRAMP vaccines demonstrated clinical benefit of treated mice and enhanced anti-tumour immune response.

Whole cell melanoma vaccine genetically modified to stem cells like phenotype generates specific immune responses to ALDH1A1 and long-term survival in advanced melanoma patients

ABSTRACT Allogeneic whole cell gene modified therapeutic melanoma vaccine (AGI-101H) comprising of two melanoma cell lines transduced with cDNA encoding fusion protein composed of IL-6 linked with the soluble IL-6 receptor (sIL-6R), referred to as H6 was developed. H6 served as a molecular adjuvant, however, it has altered vaccine cells phenotype towards melanoma stem cells (MSC)-like with high activity of aldehyde dehydrogenase isoenzyme (ALDH1A1). AGI-101H was applied in advanced melanoma patients with non-resected and resected disease. In the adjuvant setting, it was combined with surgery in case of recurring metastases, which were surgically removed and vaccination continued. A significant fraction of AGI-101H treated melanoma patients is still alive (11–19 years). Out of 106 living patients, 39 were HLA-A2 positive and were the subject of the study. Immunization of melanoma patients resulted in the generation of cytotoxic CD8+ T cells specific for ALDH1A1, which were detected in circulation by HLA-A0201 MHC dextramers loaded with ALDH1A188-96(LLYKLADLI) peptide. Phenotypically they were central memory CD8+ T cells. Re-stimulation with ALDH1A188-96 ex vivo resulted in IFN-γ secretion and cells degranulation. Following each vaccine dose administration, the number of ALDH1A1-CD8+ T cells increased in circulation and returned to the previous level until next dose injection (one month). ALDH1A1-CD8+ T cells were also found, however in the lower number than in vaccinated patients, in the circulation of untreated melanoma with stage IV but were not found in stage II or III and healthy donors. Specific anti-ALDH1 antibodies were present in treated patients. Long-term survival suggests immuno-targeting of MSC in treated patients.