Stacey L. Lehman

ER stress–mediated autophagy promotes Myc-dependent transformation and tumor growth

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

ATF4-dependent induction of heme oxygenase 1 prevents anoikis and promotes metastasis

The integrated stress response (ISR) is a critical mediator of cancer cell survival, and targeting the ISR inhibits tumor progression. Here, we have shown that activating transcription factor 4 (ATF4), a master transcriptional effector of the ISR, protects transformed cells against anoikis - a specialized form of apoptosis - following matrix detachment and also contributes to tumor metastatic properties. Upon loss of attachment, ATF4 activated a coordinated program of cytoprotective autophagy and antioxidant responses, including induced expression of the major antioxidant enzyme heme oxygenase 1 (HO-1). HO-1 upregulation was the result of simultaneous activation of ATF4 and the transcription factor NRF2, which converged on the HO1 promoter. Increased levels of HO-1 ameliorated oxidative stress and cell death. ATF4-deficient human fibrosarcoma cells were unable to colonize the lungs in a murine model, and reconstitution of ATF4 or HO-1 expression in ATF4-deficient cells blocked anoikis and rescued tumor lung colonization. HO-1 expression was higher in human primary and metastatic tumors compared with noncancerous tissue. Moreover, HO-1 expression correlated with reduced overall survival of patients with lung adenocarcinoma and glioblastoma. These results establish HO-1 as a mediator of ATF4-dependent anoikis resistance and tumor metastasis and suggest ATF4 and HO-1 as potential targets for therapeutic intervention in solid tumors.

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O2 for 8 h only (O2), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O2 + IR) followed by 16 h of normoxia (ambient air containing 21% O2 and 5% CO2) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O2 + IR exacerbated cell death and DNA damage compared to individual exposures O2 or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest.

Signaling through alternative Integrated Stress Response pathways compensates for GCN2 loss in a mouse model of soft tissue sarcoma

The tumor microenvironment is characterized by deficiencies in oxygen and nutrients, such as glucose and amino acids. Activation of the GCN2 arm of the Integrated Stress Response (ISR) in response to amino acid deprivation is one mechanism by which tumor cells cope with nutrient stress. GCN2 phosphorylates the alpha subunit of the eukaryotic translation initiation factor eIF2, leading to global downregulation of translation to conserve amino acids and initiation of a transcriptional program through ATF4 to promote recovery from nutrient deprivation. Loss of GCN2 results in decreased tumor cell survival in vitro under amino acid deprivation and attenuated tumor growth in xenograft tumor models. However, it is not known what effects GCN2 loss has on the growth of autochthonous tumors that arise in their native microenvironment. Here, we demonstrate in a genetically engineered mouse model of soft tissue sarcoma that loss of GCN2 has no effect on tumor growth or animal survival. The sarcomas displayed compensatory activation of PERK or phospho-eIF2α independent upregulation of ATF4 in order to maintain ISR signaling, indicating that this pathway is critical for tumorigenesis. These results have important implications for the development and testing of small molecule inhibitors of ISR kinases as cancer therapeutics.

Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress

Multiple transcripts encode for the cell cycle inhibitor p21Cip1. These transcripts produce identical proteins but differ in their 5’ untranslated regions (UTRs). Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through 35S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR) kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5’ upstream open reading frames (uORFs) through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.

Radiation-induced translational control of gene expression

ABSTRACT Radiation-induced gene expression has long been hypothesized to protect against cell death. Defining this process would provide not only insight into the mechanisms mediating cell survival after radiation exposure, but also a novel source of targets for radiosensitization. However, whereas the radiation-induced gene expression profiles using total cellular mRNA have been generated for cell lines as well as normal tissues, with few exception, the changes in mRNA do not correlate with changes in the corresponding protein. The traditional approach to profiling gene expression, i.e., using total cellular RNA, does not take into account posttranscriptional regulation. In this review, we describe the use of gene expression profiling of polysome-bound RNA to establish that radiation modifies gene expression via translational control. Because changes in polysome-bound mRNA correlate with changes in protein, analysis of the translational profiles provides a unique data set for investigating the mechanisms mediating cellular radioresponse.

The role of autophagy as a mechanism of cytotoxicity by the clinically used agent MDA-7/IL-24.

Commentary to: OSU-03012 enhances Ad.7-induced GBM cell killing via ER stress and autophagy and by decreasing expression of mitochondrial protective proteins Hossein A. Hamed, Adly Yacoub, Margaret A. Park, Patrick Eulitt, Devanand Sarkar, Igor P. Dimitrie, Ching-Shih Chen, Steven Grant, David T. Curiel, Paul B. Fisher and Paul Dent

Abstract LB-175: ATM regulates radiation-induced translational control of gene expression

The cellular radioresponse is composed of processes regulated by constitutively expressed proteins and processes regulated by new gene expression. While processes regulated by constitutively expressed proteins, such as those participating in DNA repair and cell cycle arrest, have been extensively studied, the radiation-induced changes in gene expression are not well defined. Previously, we have demonstrated that translation plays an important role in regulating gene expression in response to radiation. In glioblastoma cells, treatment with radiation affects more genes at the translational level than at the transcriptional level. Specific transcripts were found to be recruited to or away from polysomes without global changes in the polysome profile of the cells. Here, we utilized microarray analysis of polysome-associated transcripts in the glioblastoma stem-like cell line NSC11 to demonstrate that the DNA damage response kinase ATM controls the translation of a subset of genes as part of the radioresponse. Cells were incubated with the ATM inhibitor KU-60019 at a dose of 500nM for one hour, exposed to 2 Gy, and harvested for polysome extraction one hour after irradiation. ATM inhibition blocked translational changes of approximately one third of genes that were affected by radiation. Gene enrichment analysis revealed that genes translationally regulated by ATM were significantly involved in a number of cellular processes, indicating that ATM-mediated translational control may have functional consequences on the cellular radioresponse. By GSEA, cells treated with radiation alone were highly enriched for terms involving mitochondrial structure and function, cellular respiration, and oxidative stress as compared to irradiated cells treated with the ATM inhibitor. Other significantly enriched categories of functional interest included the spliceosome, RNA catabolic processes, the small nuclear ribonucleoprotein complex, and phospholipid and phosphoinositide metabolism. Analysis of UTR regulatory elements revealed that a significant percentage of the genes translationally upregulated by ATM in irradiated cells were enriched for HuR binding sites, which suggests a potential mechanism for polysome recruitment of these transcripts. Overall, these findings uncover a novel network of post-transcriptional gene regulation orchestrated by ATM and provide a framework to identify potential new molecules and pathways that govern tumor cell radiosensitivity. Citation Format: Stacey L. Lehman, Amy Wahba, Kevin Camphausen, Philip J. Tofilon. ATM regulates radiation-induced translational control of gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-175.

Abstract 1262: The transcription factor ATF4 regulates resistance to anoikis and promotes metastasis in fibrosarcoma via cooperative upregulation of Heme Oxygenase-1 with Nrf2

The Integrated Stress Response (ISR) plays a critical role in cancer cell survival, and targeting the ISR results in inhibition of tumor progression. A critical aspect of ISR involves the preferential translation of activating transcription factor 4(ATF4), a transcriptional factor regulating genes involved in metabolism, nutrient uptake, and anti-oxidant responses. We previously demonstrated that ATF4 expression is significantly increased in tumors compared to corresponding normal tissue, and that ablation of ATF4 compromised primary tumor growth in mice. Based on the central roles that ATF4 target genes play in pro-survival processes, we hypothesized that ATF4 might also play a role in tumor metastasis. Upon loss of matrix attachment, a critical step in the metastatic process, we found induction of phosphorylation of the translation factor eIF2α mediated by upstream kinase PERK in human adenocarcinoma HT1080 cells and colorectal adenocarcinoma DLD1 cells. Increased eIF2α phosphorylation resulted in translational upregulation of ATF4 and its transcriptional targets CHOP, ASNS and ATF3. Interestingly, failure to induce ISR and upregulate ATF4 resulted in increased apoptosis following matrix detachment - a process known as anoikis. Furthermore, we demonstrate that ATF4 promotes anoikis resistance by activating a coordinated program of autophagy and anti-oxidant responses. Upon detachment, ATF4 activates cytoprotective autophagy by transcriptionally upregulating several key autophagic genes (Atg5, Atg7 and Ulk1). Simultaneously, ATF4 also induces the expression of the heme oxygenase 1 (HO-1) - a major antioxidant enzyme. Activation of HO-1 following matrix detachment occurs by coordinated upregulation of ATF4 and the antioxidant PERK dependent transcription factor Nrf2, which converge to bind on antioxidant regulatory elements (ARE) in the HO-1 promoter. Failure to either initiate the autophagy response or HO-1 induction sensitizes cells to anoikis. In agreement with our in vitro observations, HT1080 cells harboring ATF4 shRNA (shATF4) injected through tail vein of nude mice fails to establish metastatic lung colonization after 4 weeks compared to the non-targeting (shNT) counterparts. Immunohistochemical analysis on the tumor bearing lungs show high and colocalized expression of ATF4 and HO-1. Reconstituting either ATF4 or HO-1 expression in ATF4-deficient cells, rescues the tumor lung colonization phenotype. Finally, higher expression of HO-1 and ATF4 was found in human primary as well as metastatic tumors compared to normal epithelium or stromal tissue and correlated with reduced overall survival of lung adenocarcinoma and glioblastoma patients. Collectively, these results establish ATF4 as a major player in tumor metastasis, and the combined activity of its downstream targets HO-1 and Nrf2 as a critical mediator in this process. Citation Format: Souvik Dey, Carly M. Sayers, Stacey L. Lehman, Yi Cheng, George J. Cerniglia, Stephen W. Tuttle, Michael D. Feldman, Paul J.L. Zhang, Serge Y. Fuchs, J. Alan Diehl, Constantinos Koumenis. The transcription factor ATF4 regulates resistance to anoikis and promotes metastasis in fibrosarcoma via cooperative upregulation of Heme Oxygenase-1 with Nrf2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1262. doi:10.1158/1538-7445.AM2015-1262

The Unfolded Protein Response and Therapeutic Opportunities

Tumor cells employ multiple elaborate, evolutionarily conserved mechanisms that enable them to respond to stress conditions in the tumor microenvironment, including hypoxia. Although the most studied cellular signaling pathway induced by hypoxia is mediated by the transcriptional activity of hypoxia-inducible factors (HIFs), several HIF-independent mechanisms have been implicated in hypoxic adaptation, especially in the regulation of macromolecular synthesis. One such mechanism, known as the unfolded protein response (UPR), encompasses a trio of cellular signaling cascades. The UPR is activated by the accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER). Numerous in vitro and in vivo studies using genetic and pharmacological modifications of UPR signaling components have demonstrated an important role for the UPR in determining tumor cell survival following transient and chronic hypoxia. This review summarizes the important aspects of UPR signaling and the role of the UPR in determining tumor cell survival or death under hypoxic stress. We also discuss novel pharmacological approaches for targeting critical UPR components as potential anti-tumor strategies.