The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways - cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.

Signal integration in the endoplasmic reticulum unfolded protein response

Interest in the topic of tumour metabolism has waxed and waned over the past century of cancer research. The early observations of Warburg and his contemporaries established that there are fundamental differences in the central metabolic pathways operating in malignant tissue. However, the initial hypotheses that were based on these observations proved inadequate to explain tumorigenesis, and the oncogene revolution pushed tumour metabolism to the margins of cancer research. In recent years, interest has been renewed as it has become clear that many of the signalling pathways that are affected by genetic mutations and the tumour microenvironment have a profound effect on core metabolism, making this topic once again one of the most intense areas of research in cancer biology.

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Regulation of cancer cell metabolism

ER stress-induced cell death mechanisms

Cells respond to stress stimuli through coordinated changes in gene expression. The regulation of translation is often used under these circumstances because it allows immediate and selective changes in protein levels. There are many examples of translational control in response to stress. Here we examine two representative models, the regulation of eukaryotic initiation factor-2alpha by phosphorylation and internal ribosome initiation through the internal ribosome-entry site, which illustrate the importance of translational control in the cellular stress response and apoptosis.

Translational control in stress and apoptosis

Obesity is linked to a variety of metabolic disorders, such as insulin resistance and atherosclerosis. Dysregulated production of fat-derived secretory factors, adipocytokines, is partly responsible for obesity-linked metabolic disorders. However, the mechanistic role of obesity per se to adipocytokine dysregulation has not been fully elucidated. Here, we show that adipose tissue of obese mice is hypoxic and that local adipose tissue hypoxia dysregulates the production of adipocytokines. Tissue hypoxia was confirmed by an exogenous marker, pimonidazole, and by an elevated concentration of lactate, an endogenous marker. Moreover, local tissue hypoperfusion (measured by colored microspheres) was confirmed in adipose tissue of obese mice. Adiponectin mRNA expression was decreased, and mRNA of C/EBP homologous protein (CHOP), an endoplasmic reticulum (ER) stress-mediated protein, was significantly increased in adipose tissue of obese mice. In 3T3-L1 adipocytes, hypoxia dysregulated the expression of adipocytokines, such as adiponectin and plasminogen activator inhibitor type-1, and increased the mRNAs of ER stress marker genes, CHOP and GRP78 (glucose-regulated protein, 78 kD). Expression of CHOP attenuated adiponectin promoter activity, and RNA interference of CHOP partly reversed hypoxia-induced suppression of adiponectin mRNA expression in adipocytes. Hypoxia also increased instability of adiponectin mRNA. Our results suggest that hypoperfusion and hypoxia in adipose tissues underlie the dysregulated production of adipocytokines and metabolic syndrome in obesity.

https://diabetes.diabetesjournals.org/content/diabetes/56/4/901.full.pdf

Adipose Tissue Hypoxia in Obesity and Its Impact on Adipocytokine Dysregulation

Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.

Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase perk and phosphorylation of the translation initiation factor eif2alpha

Activation of the epidermal growth factor receptor (EGFR) and downstream signaling pathways, such as phosphatidylinositol-3 kinase/Akt and Ras/mitogen-activated protein kinase (MAPK), have been implicated in causing resistance to EGFR-targeted therapy in solid tumors, including the urogenital tumors. To investigate the mechanism of resistance to EGFR inhibition in bladder cancer, we compared EGFR tyrosine kinase inhibitor (Gefitinib, Iressa, ZD1839) with respect to its inhibitory effects on three kinases situated downstream of EGFR: MAPK, Akt, and glycogen synthase kinase-3β (GSK-3β). We found that the resistance to the antiproliferative effects of gefitinib, in vitro as well as in vivo in nude mice models, was associated with uncoupling between EGFR and MAPK inhibition, and that GSK-3β activation and degradation of its target cyclin D1 were indicators of a high cell sensitivity to gefitinib. Further analysis of one phenotypic sensitive (253J B-V) and resistant (UM-UC13) cell lines revealed that platelet-derived growth factor receptor-β (PDGFRβ) activation was responsible for short circuiting the EGFR/MAPK pathway for mitogenic stimuli. However, invasion as well as actin dynamics were efficiently reduced by EGFR inhibition in UM-UC13. Chemical disruption of signaling pathways or of PDGFR kinase activity significantly reduced the inactive pool of cellular GSK-3β in UM-UC13 cells. In conclusion, our data show that the uncoupling of EGFR with mitogenic pathways can cause resistance to EGFR inhibition in bladder cancer. Although this uncoupling may arise through different mechanisms, we suggest that the resistance of bladder cancer cells to EGFR blockade can be predicted early in the course of treatment by measuring the activation of GSK-3β and of nuclear cyclin D1.

https://cancerres.aacrjournals.org/content/canres/65/22/10524.full.pdf

Uncoupling between Epidermal Growth Factor Receptor and Downstream Signals Defines Resistance to the Antiproliferative Effect of Gefitinib in Bladder Cancer Cells

Abstract 1254: Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations


 Despite recent advances in prevention and treatment, including immune checkpoint inhibitors, malignant melanoma remains a particularly aggressive and deadly malignancy, which is partly attributed to its highly heterogeneous TME. However, malignant cells exhibit altered signaling pathways, which enables them to adapt to both cell-intrinsic and extrinsic stressors within TME. The activating transcription factor 4 (ATF4) is a master transcriptional effector of the Integrated Stress Response (ISR), a homeostatic mechanism coupling cell growth and survival to bioenergetic demands. We and others have established a critical tumor cell-intrinsic role of ATF4 which culminates in the promotion of primary tumor growth and in the establishment of micro- and macro-metastases in xenograft, allograft and transgenic models. However, the potential roles of the ISR and particularly of ATF4-mediated responses in host-dependent, tumor-related processes, have not been yet extensively investigated. Using novel conditional knockout ATF4 mouse models, we show that global loss of host ATF4 results in deficient tumor vascularization and a pronounced tumor growth delay in syngeneic melanoma and pancreatic tumor models. Immunofluorescence analysis revealed a severely impaired angiogenic phenotype in tumors grown in ATF4 KO mice which was accompanied by deficiencies in markers of CAF activation. Single-cell transcriptomic analysis of B16F10 melanoma tumors further localized this defect to a distinct CAF population, previously identified as vascular CAFs (vCAFs), and revealed a significant reduction in the expression of extracellular matrix components, primarily type I collagen, in tumors grown in ATF4 KO mice. Intriguingly, we identified a multifaceted impairment of the collagen biosynthetic pathway with the ATF4 to directly regulate the expression of the Col1a1 gene as well as the intracellular levels of glycine and proline, the major amino acids comprising collagen fibers. Moreover, we showed that the ATF4-deficient vCAFs secrete significantly lower levels of angiogenic factors (i.e., VEGF, SDF-1 etc.) in the perivascular area leading to an abnormal angiogenesis and significant attenuation of tumor growth. Specific deletion of ATF4 in the fibroblast compartment (Col1a1 promoter) produced a similar tumor growth delay as in the global ATF4 KO mice, and notably, co-injection of fibroblasts from ATF4-proficient mice led to significant recovery of tumor growth rates in ATF4-deficient mice. Finally, analysis of human melanoma and pancreatic tumor samples revealed a strong correlation between ATF4 and collagen levels and between an ISR gene signature and expression of collagen and CAF activation genes. Our findings uncover a novel role of stromal ATF4 in shaping CAF functionality, a key driver of disease progression, metastasis, and therapy resistance.
 Citation Format: Ioannis I. Verginadis, Harris Avgousti, Kyle Kim, Giorgos Skoufos, Frank Chinga, Nektaria Maria Leli, Ilias V. Karagounis, Brett I. Bell, Anastasia Velalopoulou, Victoria S. Wu, Yang Li, Jiangbin Ye, David A. Scott, Andrei L. Osterman, Arjun Sengupta, Aalim Weljie, Artemis G. Hatzigeorgiou, Sandra Ryeom, Alan J. Diehl, Serge Y. Fuchs, Ellen Puré, Constantinos Koumenis. A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3178.

Alan J. Diehl

Papers

Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase perk and phosphorylation of the translation initiation factor eif2alpha

Uncoupling between Epidermal Growth Factor Receptor and Downstream Signals Defines Resistance to the Antiproliferative Effect of Gefitinib in Bladder Cancer Cells

Abstract 1254: Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations

Abstract 3178: A stromal integrated stress response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumor progression