miR-217 Regulates Normal and Tumor Cell Fate Following Induction of Endoplasmic Reticulum Stress.

Rapidly proliferating cancer cells require a microenvironment where essential metabolic nutrients like glucose, oxygen, and growth factors become scarce as the tumor volume surpasses the established vascular capacity of the tissue. Limits in nutrient availability typically trigger growth arrest and/or apoptosis to prevent cellular expansion. However, tumor cells frequently co-opt cellular survival pathways thereby favoring cell survival under this environmental stress. The unfolded protein response (UPR) pathway is typically engaged by tumor cells to favor adaptation to stress. PERK, an endoplasmic reticulum (ER) protein kinase and UPR effector is activated in tumor cells and contributes tumor cell adaptation by limiting protein translation and balancing redox stress. PERK also induces miRNAs that contribute to tumor adaptation. miR-211 and miR-216b were previously identified as PERK-ATF4-regulated miRNAs that regulate cell survival. We have identified another PERK-responsive miRNA, miR-217, with increased expression under prolonged ER stress. Key targets of miR-217 are identified as TRPM1, the host gene for miR-211 and EZH2. Evidence is provided that miR-217 expression is essential for the rapid loss of miR-211 in prolonged ER stress and provides a functional link for determining whether cells adapt to stress or commit to apoptosis. IMPLICATIONS: PERK-dependent induction of miR-217 limits accumulation and function of the prosurvival miRNA, miR-211, to establish cell fate and promote cell commitment to apoptosis.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming. One-Sentence Summary: After severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

A phenomenological model of proton FLASH oxygen depletion effects depending on tissue vasculature and oxygen supply.

Introduction: Radiation-induced oxygen depletion in tissue is assumed as a contributor to the FLASH sparing effects. In this study, we simulated the heterogeneous oxygen depletion in the tissue surrounding the vessels and calculated the proton FLASH effective-dose-modifying factor (FEDMF), which could be used for biology-based treatment planning. Methods: The dose and dose-weighted linear energy transfer (LET) of a small animal proton irradiator was simulated with Monte Carlo simulation. We deployed a parabolic partial differential equation to account for the generalized radiation oxygen depletion, tissue oxygen diffusion, and metabolic processes to investigate oxygen distribution in 1D, 2D, and 3D solution space. Dose and dose rates, particle LET, vasculature spacing, and blood oxygen supplies were considered. Using a similar framework for the hypoxic reduction factor (HRF) developed previously, the FEDMF was derived as the ratio of the cumulative normoxic-equivalent dose (CNED) between CONV and UHDR deliveries. Results: Dynamic equilibrium between oxygen diffusion and tissue metabolism can result in tissue hypoxia. The hypoxic region displayed enhanced radio-resistance and resulted in lower CNED under UHDR deliveries. In 1D solution, comparing 15 Gy proton dose delivered at CONV 0.5 and UHDR 125 Gy/s, 61.5% of the tissue exhibited ≥20% FEDMF at 175 µm vasculature spacing and 18.9 µM boundary condition. This percentage reduced to 34.5% and 0% for 8 and 2 Gy deliveries, respectively. Similar trends were observed in the 3D solution space. The FLASH versus CONV differential effect remained at larger vasculature spacings. A higher FLASH dose rate showed an increased region with ≥20% FEDMF. A higher LET near the proton Bragg peak region did not appear to alter the FLASH effect. Conclusion: We developed 1D, 2D, and 3D oxygen depletion simulation process to obtain the dynamic HRF and derive the proton FEDMF related to the dose delivery parameters and the local tissue vasculature information. The phenomenological model can be used to simulate or predict FLASH effects based on tissue vasculature and oxygen concentration data obtained from other experiments.

Clinical measures, radiomics, and genomics offer synergistic value in AI-based prediction of overall survival in patients with glioblastoma.

Multi-omic data, i.e., clinical measures, radiomic, and genetic data, capture multi-faceted tumor characteristics, contributing to a comprehensive patient risk assessment. Here, we investigate the additive value and independent reproducibility of integrated diagnostics in prediction of overall survival (OS) in isocitrate dehydrogenase (IDH)-wildtype GBM patients, by combining conventional and deep learning methods. Conventional radiomics and deep learning features were extracted from pre-operative multi-parametric MRI of 516 GBM patients. Support vector machine (SVM) classifiers were trained on the radiomic features in the discovery cohort (n = 404) to categorize patient groups of high-risk (OS < 6 months) vs all, and low-risk (OS ≥ 18 months) vs all. The trained radiomic model was independently tested in the replication cohort (n = 112) and a patient-wise survival prediction index was produced. Multivariate Cox-PH models were generated for the replication cohort, first based on clinical measures solely, and then by layering on radiomics and molecular information. Evaluation of the high-risk and low-risk classifiers in the discovery/replication cohorts revealed area under the ROC curves (AUCs) of 0.78 (95% CI 0.70-0.85)/0.75 (95% CI 0.64-0.79) and 0.75 (95% CI 0.65-0.84)/0.63 (95% CI 0.52-0.71), respectively. Cox-PH modeling showed a concordance index of 0.65 (95% CI 0.6-0.7) for clinical data improving to 0.75 (95% CI 0.72-0.79) for the combination of all omics. This study signifies the value of integrated diagnostics for improved prediction of OS in GBM.

Comparison of FLASH Proton Entrance and the Spread-Out Bragg Peak Dose Regions in the Sparing of Mouse Intestinal Crypts and in a Pancreatic Tumor Model.

Ultra-high dose rate FLASH proton radiotherapy (F-PRT) has been shown to reduce normal tissue toxicity compared to standard dose rate proton radiotherapy (S-PRT) in experiments using the entrance portion of the proton depth dose profile, while proton therapy uses a spread-out Bragg peak (SOBP) with unknown effects on FLASH toxicity sparing. To investigate, the biological effects of F-PRT using an SOBP and the entrance region were compared to S-PRT in mouse intestine. In this study, 8-10-week-old C57BL/6J mice underwent 15 Gy (absorbed dose) whole abdomen irradiation in four groups: (1) SOBP F-PRT, (2) SOBP S-PRT, (3) entrance F-PRT, and (4) entrance S-PRT. Mice were injected with EdU 3.5 days after irradiation, and jejunum segments were harvested and preserved. EdU-positive proliferating cells and regenerated intestinal crypts were quantified. The SOBP had a modulation (width) of 2.5 cm from the proximal to distal 90%. Dose rates with a SOBP for F-PRT or S-PRT were 108.2 ± 8.3 Gy/s or 0.82 ± 0.14 Gy/s, respectively. In the entrance region, dose rates were 107.1 ± 15.2 Gy/s and 0.83 ± 0.19 Gy/s, respectively. Both entrance and SOBP F-PRT preserved a significantly higher number of EdU + /crypt cells and percentage of regenerated crypts compared to S-PRT. Moreover, tumor growth studies showed no difference between SOBP and entrance for either of the treatment modalities.

Current delivery limitations of proton PBS for FLASH.

PURPOSE: Proton Pencil Beam Scanning (PBS) is an attractive solution to realize the advantageous normal tissue sparing elucidated from FLASH high dose rates. The mechanics of PBS spot delivery will impose limitations on the effective field dose rate for PBS. METHODS: This study incorporates measurements from clinical and FLASH research beams on uniform single energy and the spread-out Bragg Peak PBS fields to extrapolate the PBS dose rate to high cyclotron beam currents 350, 500, and 800 nA. The impact of the effective field dose rate from cyclotron current, spot spacing, slew time and field size were studied. RESULTS: When scanning magnet slew time and energy switching time are not considered, single energy effective field FLASH dose rate (≥40 Gy/s) can only be achieved with less than 4 × 4 cm2 fields when the cyclotron output current is above 500 nA. Slew time and energy switching time remain the limiting factors for achieving high effective dose rate of the field. The dose rate-time structures were obtained. The amount of the total dose delivered at the FLASH dose rate in single energy layer and volumetric field was also studied. CONCLUSION: It is demonstrated that while it is difficult to achieve FLASH dose rate for a large field or in a volume, local FLASH delivery to certain percentage of the total dose is possible. With further understanding of the FLASH radiobiological mechanism, this study could provide guidance to adapt current clinical multi-field proton PBS delivery practice for FLASH proton radiotherapy.

Design, Implementation, and in Vivo Validation of a Novel Proton FLASH Radiation Therapy System.

PURPOSE: Recent studies suggest that ultrahigh-dose-rate, "FLASH," electron radiation therapy (RT) decreases normal tissue damage while maintaining tumor response compared with conventional dose rate RT. Here, we describe a novel RT apparatus that delivers FLASH proton RT (PRT) using double scattered protons with computed tomography guidance and provide the first report of proton FLASH RT-mediated normal tissue radioprotection. METHODS AND MATERIALS: Absolute dose was measured at multiple depths in solid water and validated against an absolute integral charge measurement using a Faraday cup. Real-time dose rate was obtained using a NaI detector to measure prompt gamma rays. The effect of FLASH versus standard dose rate PRT on tumors and normal tissues was measured using pancreatic flank tumors (MH641905) derived from the KPC autochthonous PanCa model in syngeneic C57BL/6J mice with analysis of fibrosis and stem cell repopulation in small intestine after abdominal irradiation. RESULTS: The double scattering and collimation apparatus was dosimetrically validated with dose rates of 78 ± 9 Gy per second and 0.9 ± 0.08 Gy per second for the FLASH and standard PRT. Whole abdominal FLASH PRT at 15 Gy significantly reduced the loss of proliferating cells in intestinal crypts compared with standard PRT. Studies with local intestinal irradiation at 18 Gy revealed a reduction to near baseline levels of intestinal fibrosis for FLASH-PRT compared with standard PRT. Despite this difference, FLASH-PRT did not demonstrate tumor radioprotection in MH641905 pancreatic cancer flank tumors after 12 or 18 Gy irradiation. CONCLUSIONS: We have designed and dosimetrically validated a FLASH-PRT system with accurate control of beam flux on a millisecond time scale and online monitoring of the integral and dose delivery time structure. Using this system, we found that FLASH-PRT decreases acute cell loss and late fibrosis after whole-abdomen and focal intestinal RT, whereas tumor growth inhibition is preserved between the 2 modalities.

miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis.

The ability of the unfolded protein response, UPR, to regulate cell homeostasis through both gene expression and protein synthesis has been well documented. One primary pro-apoptotic protein that responds to both PERK and Ire1 signalling is the CHOP/GADD153 transcription factor. Although CHOP deficiency delays onset of cell death, questions remain regarding how CHOP regulates apoptosis. Here, we provide evidence demonstrating that CHOP/GADD153-dependent apoptosis reflects expression of micro-RNA, miR-216b. MiR-216b accumulation requires PERK-dependent induction of CHOP/GADD153, which then directly regulates miR-216b expression. As maximal expression of miR-216b is antagonized by Ire1, miR-216b accumulation reflects the convergence of PERK and Ire1 activities. Functionally, miR-216b directly targets c-Jun, thereby reducing AP-1-dependent transcription and sensitizing cells to ER stress-dependent apoptosis. These results provide direct insight into the molecular mechanisms of CHOP/GADD153-dependent cell death.

MDA-7/IL-24-induced cell killing in malignant renal carcinoma cells occurs by a ceramide/CD95/PERK-dependent mechanism.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on clarifying the mechanism(s) by which glutathione S-transferase (GST)-MDA-7 altered cell survival of human renal carcinoma cells in vitro. GST-MDA-7 caused plasma membrane clustering of CD95 and the association of CD95 with procaspase-8. GST-MDA-7 lethality was suppressed by inhibition of caspase-8 or by overexpression of short-form cellular FLICE inhibitory protein, but only weakly by inhibition of cathepsin proteases. GST-MDA-7-induced CD95 clustering (and apoptosis) was blocked by knockdown of acidic sphingomyelinase or, to a greater extent, ceramide synthase-6 expression. GST-MDA-7 killing was, in parallel, dependent on inactivation of extracellular signal-regulated kinase 1/2 and on CD95-induced p38 mitogen-activated protein kinase and c-jun NH(2)-terminal kinase-1/2 signaling. Knockdown of CD95 expression abolished GST-MDA-7-induced phosphorylation of protein kinase R-like endoplasmic reticulum kinase. GST-MDA-7 lethality was suppressed by knockout or expression of a dominant negative protein kinase R-like endoplasmic reticulum kinase that correlated with reduced c-jun NH(2)-terminal kinase-1/2 and p38 mitogen-activated protein kinase signaling and maintained extracellular signal-regulated kinase-1/2 phosphorylation. GST-MDA-7 caused vacuolization of LC3 through a mechanism that was largely CD95 dependent and whose formation was suppressed by knockdown of ATG5 expression. Knockdown of ATG5 suppressed GST-MDA-7 toxicity. Our data show that in kidney cancer cells GST-MDA-7 induces ceramide-dependent activation of CD95, which is causal in promoting an endoplasmic reticulum stress response that activates multiple proapoptotic pathways to decrease survival.

PERK-dependent regulation of MDA-7/IL-24-induced autophagy in primary human glioma cells.

Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The studies by Yacoub et al. (Mol Cancer Ther 2008; 7:314-29) further defines the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that were dependent on activation of JNK1-3 with subsequent activation of BAX and the induction of mitochondrial dysfunction. Activation of JNK1-3 was dependent upon protein kinase R-like endoplasmic reticulum kinase (PERK) and GST-MDA-7 lethality was suppressed in PERK(-/-) cells. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or of BiP/GRP78, or by knockdown of ATG5 or Beclin 1 expression, but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin 1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data demonstrate that GST-MDA-7 induces an ER stress response that, via the induction of autophagy, is causal in the activation of pro-apoptotic pathways that converge on the mitochondrion and ultimately culminate in decreased glioma cell survival.

Caspase-, cathepsin-, and PERK-dependent regulation of MDA-7/IL-24-induced cell killing in primary human glioma cells.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which a GST-MDA-7 fusion protein inhibits cell survival of primary human glioma cells in vitro. GST-MDA-7 killed glioma cells with diverse genetic characteristics that correlated with inactivation of ERK1/2 and activation of JNK1-3. Activation of JNK1-3 was dependent on protein kinase R-like endoplasmic reticulum kinase (PERK), and GST-MDA-7 lethality was suppressed in PERK-/- cells. JNK1-3 signaling activated BAX, whereas inhibition of JNK1-3, deletion of BAX, or expression of dominant-negative caspase-9 suppressed lethality. GST-MDA-7 also promoted a PERK-, JNK-, and cathepsin B-dependent cleavage of BID; loss of BID function promoted survival. GST-MDA-7 suppressed BAD and BIM phosphorylation and heat shock protein 70 (HSP70) expression. GST-MDA-7 caused PERK-dependent vacuolization of LC3-expressing endosomes whose formation was suppressed by incubation with 3-methyladenine, expression of HSP70 or BiP/GRP78, or knockdown of ATG5 or Beclin-1 expression but not by inhibition of the JNK1-3 pathway. Knockdown of ATG5 or Beclin-1 expression or overexpression of HSP70 reduced GST-MDA-7 lethality. Our data show that GST-MDA-7 induces an endoplasmic reticulum stress response that is causal in the activation of multiple proapoptotic pathways, which converge on the mitochondrion and highlight the complexity of signaling pathways altered by mda-7/IL-24 in glioma cells that ultimately culminate in decreased tumor cell survival.

Regulation of GST-MDA-7 toxicity in human glioblastoma cells by ERBB1, ERK1/2, PI3K, and JNK1-3 pathway signaling.

The present studies defined the biological effects of a GST fusion protein of melanoma differentiation-associated gene-7 (mda-7), GST-MDA-7 (1 and 30 nmol/L), on cell survival and cell signaling in primary human glioma cells in vitro. GST-MDA-7, in a dose- and time-dependent fashion killed glioma cells with diverse genetic characteristics; 1 nmol/L caused arrest without death, whereas 30 nmol/L caused arrest and killing after exposure. Combined inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT function was required to enhance 1 nmol/L GST-MDA-7 lethality in all cell types, whereas combined activation of MEK1 and AKT was required to suppress 30 nmol/L GST-MDA-7 lethality; both effects are mediated in part by modulating c-Jun NH(2)-terminal kinase (JNK) 1-3 activity. The geldanamycin 17AAG inhibited AKT and ERK1/2 in GBM cells and enhanced GST-MDA-7 lethality. JNK1-3 signaling promoted BAX activation and mitochondrial dysfunction. In GBM6 cells, GST-MDA-7 (30 nmol/L) transiently activated p38 mitogen-activated protein kinase, which was modestly protective against JNK1-3-induced toxicity, whereas GST-MDA-7 (300 nmol/L) caused prolonged intense p38 mitogen-activated protein kinase activation, which promoted cell death. In GBM12 cells that express full-length mutant activated ERBB1, inhibition of ERBB1 did not modify GST-MDA-7 lethality; however, in U118 established glioma cells, stable overexpression of wild-type ERBB1 and/or truncated active ERBB1vIII suppressed GST-MDA-7 lethality. Our data argue that combined inhibition of ERK1/2 and AKT function, regardless of genetic background, promotes MDA-7 lethality in human primary human glioma cells via JNK1-3 signaling and is likely to represent a more ubiquitous approach to enhancing MDA-7 toxicity in this cell type than inhibition of ERBB1 function.

PERK-dependent regulation of HSP70 expression and the regulation of autophagy.

The manuscript by Park et al. (Mol. Pharm. 2008; mol.107.042697 / PMID: 18182481) further defines the mechanism(s) by which OSU-03012 (OSU) kills transformed cells. It notes that in PKR-like endoplasmic reticulum kinase null cells (PERK-/-) the lethality of OSU is attenuated. OSU enhances the expression of ATG5 in a PERK-dependent fashion and promotes the ATG5-dependent formation of vesicles containing LC3, followed by a subsequent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B is activated and released into the cytosol, and genetic suppression of cathepsin B or AIF function significantly suppresses cell killing. In parallel, OSU causes PERK-dependent increases in HSP70 expression and decreases in HSP90 and Grp78/BiP expression. Inhibition of HSP70 expression enhances OSU toxicity and over-expression of HSP70 suppresses OSU-induced low pH vesicle formation and lethality. Thus, in this system PERK signaling promotes autophagy, which is causally linked to lysosomal dysfunction, cathepsin activation and cell death. However, in parallel, PERK signaling acts to suppress autophagy and lysosomal dysfunction by increasing the expression of HSP70. These findings may help explain why, in a cell type and stimulus-dependent fashion; autophagy has been noted to act either as a protective or as a toxic signal in cells.

OSU-03012 stimulates PKR-like endoplasmic reticulum-dependent increases in 70-kDa heat shock protein expression, attenuating its lethal actions in transformed cells.

We have further defined mechanism(s) by which 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide [OSU-03012 (OSU)], a derivative of the cyclooxygenase-2 (COX2) inhibitor celecoxib but lacking COX2 inhibitory activity, kills transformed cells. In cells lacking expression of protein kinase R-like endoplasmic reticulum kinase (PERK(-/-)), the lethality of OSU was attenuated. OSU enhanced the expression of Beclin 1 and ATG5 and cleavage of pro-caspase 4 in a PERK-dependent fashion and promoted the Beclin 1- and ATG5-dependent formation of vacuoles containing LC3, followed by a subsequent caspase 4-dependent cleavage of cathepsin B and a cathepsin B-dependent formation of low pH intracellular vesicles; cathepsin B was activated and released into the cytosol and genetic suppression of caspase 4, cathepsin B, or apoptosis-inducing factor function significantly suppressed cell killing. In parallel, OSU caused PERK-dependent increases in 70-kDa heat shock protein (HSP70) expression and decreases in 90-kDa heat shock protein (HSP90) and Grp78/BiP expression. Changes in HSP70 expression were post-transcriptional. Knock-down or small-molecule inhibition of HSP70 expression enhanced OSU toxicity, and overexpression of HSP70 suppressed OSU-induced low pH vesicle formation and lethality. Our data demonstrate that OSU-03012 causes cell killing that is dependent on PERK-induced activation of multiple toxic proteases. OSU-03012 also increased expression of HSP70 in a PERK-dependent fashion, providing support for the contention that OSU-03012-induced PERK signaling promotes both cell survival and cell death processes.

Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress.

It has been well established that the tumor microenvironment can promote tumor cell adaptation and survival. However, the mechanisms that influence malignant progression have not been clearly elucidated. We have previously demonstrated that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR). Here, we show that tumors derived from K-Ras-transformed Perk(-/-) mouse embryonic fibroblasts (MEFs) are smaller and exhibit less angiogenesis than tumors with an intact ISR. Furthermore, Perk promotes a tumor microenvironment that favors the formation of functional microvessels. These observations were corroborated by a microarray analysis of polysome-bound RNA in aerobic and hypoxic Perk(+/+) and Perk(-/-) MEFs. This analysis revealed that a subset of proangiogenic transcripts is preferentially translated in a Perk-dependent manner; these transcripts include VCIP, an adhesion molecule that promotes cellular adhesion, integrin binding, and capillary morphogenesis. Taken with the concomitant Perk-dependent translational induction of additional proangiogenic genes identified by our microarray analysis, this study suggests that Perk plays a role in tumor cell adaptation to hypoxic stress by regulating the translation of angiogenic factors necessary for the development of functional microvessels and further supports the contention that the Perk pathway could be an attractive target for novel antitumor modalities.

OSU-03012 promotes caspase-independent but PERK-, cathepsin B-, BID-, and AIF-dependent killing of transformed cells.

We determined one mechanism by which the putative phosphoinositide-dependent kinase (PDK)-1 inhibitor 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide (OSU-03012) killed primary human glioma and other transformed cells. OSU-03012 caused a dose-dependent induction of cell death that was not altered by p53 mutation, expression of ERBB1 vIII, or loss of phosphatase and tensin homolog deleted on chromosome 10 function. OSU-03012 promoted cell killing to a greater extent in glioma cells than in nontransformed astrocytes. OSU-03012 and ionizing radiation caused an additive, caspase-independent elevation in cell killing in 96-h viability assays and true radiosensitization in colony formation assays. In a cell type-specific manner, combined exposure to OSU-03012 with a mitogen-activated protein kinase kinase 1/2 inhibitor, phosphoinositide 3-kinase/AKT inhibitors, or parallel molecular interventions resulted in a greater than additive induction of cell killing that was independent of AKT activity and caspase function. OSU-03012 lethality as a single agent or when combined with signaling modulators was not modified in cells lacking expression of BIM or of BAX/BAK. OSU-03012 promoted the release of cathepsin B from the lysosomal compartment and release of AIF from mitochondria. Loss of BH3-interacting domain (BID) function, overexpression of BCL(XL), and inhibition of cathepsin B function suppressed cell killing and apoptosis-inducing factor (AIF) release from mitochondria. In protein kinase R-like endoplasmic reticulum kinase-/- cells, the lethality of OSU-03012 was attenuated which correlated with reduced cleavage of BID and with suppression of cathepsin B and AIF release into the cytosol. Our data demonstrate that OSU-03012 promotes glioma cell killing that is dependent on endoplasmic reticulum stress, lysosomal dysfunction, and BID-dependent release of AIF from mitochondria, and whose lethality is enhanced by irradiation or by inhibition of protective signaling pathways.

Activating transcription factor 4 is translationally regulated by hypoxic stress.

Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2alpha at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2alpha was able to attenuate the phosphorylation of eIF2alpha and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2alpha-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress.

Replication and cytopathic effect of oncolytic vesicular stomatitis virus in hypoxic tumor cells in vitro and in vivo.

Tumor hypoxia presents an obstacle to the effectiveness of most antitumor therapies, including treatment with oncolytic viruses. In particular, an oncolytic virus must be resistant to the inhibition of DNA, RNA, and protein synthesis that occurs during hypoxic stress. Here we show that vesicular stomatitis virus (VSV), an oncolytic RNA virus, is capable of replication under hypoxic conditions. In cells undergoing hypoxic stress, VSV infection produced larger amounts of mRNA than under normoxic conditions. However, translation of these mRNAs was reduced at earlier times postinfection in hypoxia-adapted cells than in normoxic cells. At later times postinfection, VSV overcame a hypoxia-associated increase in alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation and initial suppression of viral protein synthesis in hypoxic cells to produce large amounts of viral protein. VSV infection caused the dephosphorylation of the translation initiation factor eIF-4E and inhibited host translation similarly under both normoxic and hypoxic conditions. VSV produced progeny virus to similar levels in hypoxic and normoxic cells and showed the ability to expand from an initial infection of 1% of hypoxic cells to spread through an entire population. In all cases, virus infection induced classical cytopathic effects and apoptotic cell death. When VSV was used to treat tumors established in nude mice, we found VSV replication in hypoxic areas of these tumors. This occurred whether the virus was administered intratumorally or intravenously. These results show for the first time that VSV has an inherent capacity for infecting and killing hypoxic cancer cells. This ability could represent a critical advantage over existing therapies in treating established tumors.

Targeting hypoxia tolerance in cancer.

Regulation of tissue oxygen homeostasis is critical for cell function, proliferation and survival. Evidence for this continues to accumulate along with our understanding of the complex oxygen-sensing pathways present within cells. Several pathophysiological disorders are associated with a loss in oxygen homeostasis, including heart disease, stroke, and cancer. The microenvironment of tumors in particular is very oxygen heterogeneous, which may explain much of our difficulty in treating cancer effectively. This is true when comparing levels of hypoxia among different patient tumors, but also within individual tumors. Accumulating evidence implicates the biological responses to hypoxia and the alterations in these pathways in cancer as important contributors to overall malignancy and treatment efficacy. This has recently prompted several investigations into the possibility of targeting treatment at the biological responses to hypoxia.

Endoplasmic reticulum stress induces p53 cytoplasmic localization and prevents p53-dependent apoptosis by a pathway involving glycogen synthase kinase-3beta.

The tumor suppressor p53, a sensor of multiple forms of cellular stress, is regulated by post-translational mechanisms to induce cell-cycle arrest, senescence, or apoptosis. We demonstrate that endoplasmic reticulum (ER) stress inhibits p53-mediated apoptosis. The mechanism of inhibition involves the increased cytoplasmic localization of p53 due to phosphorylation at serine 315 and serine 376, which is mediated by glycogen synthase kinase-3 beta (GSK-3beta). ER stress induces GSK-3beta binding to p53 in the nucleus and enhances the cytoplasmic localization of the tumor suppressor. Inhibition of apoptosis caused by ER stress requires GSK-3beta and does not occur in cells expressing p53 with mutation(s) of serine 315 and/or serine 376 to alanine(s). As a result of the increased cytoplasmic localization, ER stress prevents p53 stabilization and p53-mediated apoptosis upon DNA damage. It is concluded that inactivation of p53 is a protective mechanism utilized by cells to adapt to ER stress.