Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response.
ABSTRACT: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.
Project description:Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3beta (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.
Project description:Within solid tumor microenvironments, lactic acidosis, and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here, we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1? protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Furthermore, these data suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells.
Project description:Under hypoxic conditions, cells suppress energy-intensive mRNA translation by modulating the mammalian target of rapamycin (mTOR) and pancreatic eIF2alpha kinase (PERK) pathways. Much is known about hypoxic inhibition of mTOR activity; however, the cellular processes activating PERK remain unclear. Since hypoxia is known to increase intracellular reactive oxygen species (ROS), we hypothesized that hypoxic ROS regulate mTOR and PERK to control mRNA translation and cell survival. Our data indicate that although exogenous ROS inhibit mTOR, eIF2alpha, and eEF2, mTOR and eEF2 were largely refractory to ROS generated under moderate hypoxia (0.5% O(2)). In direct contrast, the PERK/eIF2alpha/ATF4 integrated stress response (ISR) was activated by hypoxic ROS and contributed to global protein synthesis inhibition and adaptive ATF4-mediated gene expression. The ISR as well as exogenous growth factors were critical for cell viability during extended hypoxia, since ISR inhibition decreased the viability of cells deprived of O(2) and growth factors. Collectively, our data support an important role for ROS in hypoxic cell survival. Under conditions of moderate hypoxia, ROS induce the ISR, thereby promoting energy and redox homeostasis and enhancing cellular survival.
Project description:Neovascularization is a limiting factor in tumor growth and progression. It is well known that changes in the tumor microenvironment, such as hypoxia and glucose deprivation (GD), can induce VEGF production. However, the mechanism linking GD to tumor growth and angiogenesis is unclear. We hypothesize that GD induces the angiogenic switch in tumors through activation of the unfolded protein response (UPR). We report that UPR activation in human tumors results in elevated expression of proangiogenic mediators and a concomitant decrease in angiogenesis inhibitors. cDNA microarray results showed that GD-induced UPR activation promoted upregulation of a number of proangiogenic mediators (VEGF, FGF-2, IL-6, etc.) and downregulation of several angiogenic inhibitors (THBS1, CXCL14, and CXCL10). In vitro studies revealed that partially blocking UPR signaling by silencing protein kinase RNA-like ER kinase (PERK) or activating transcription factor 4 (ATF4) significantly reduced the production of angiogenesis mediators induced by GD. However, suppressing the alpha subunit of hypoxia-inducible factors had no effect on this process. Chromatin immunoprecipitation (ChIP) confirmed binding of ATF4 to a regulatory site in the VEGF gene. In vivo results confirmed that knockdown of PERK in tumor cells slows down tumor growth and decreases tumor blood vessel density. Collectively, these results show that the PERK/ATF4 arm of UPR mediates the angiogenic switch and is a potential target for antiangiogenic cancer therapy.
Project description:Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) signaling cascade and induction of an apoptotic cell death, autophagy, oncogenesis, metastasis, and/or resistance to cancer therapies. Mechanisms underlying regulation of ER transmembrane proteins PERK, IRE1?, and ATF6?/?, and how the balance of these activities determines outcome of the activated UPR, remain largely unclear. Here, we report a novel molecule transmembrane protein 33 (TMEM33) and its actions in UPR signaling. Immunoblotting and northern blot hybridization assays were used to determine the effects of ER stress on TMEM33 expression levels in various cell lines. Transient transfections, immunofluorescence, subcellular fractionation, immunoprecipitation, and immunoblotting were used to study the subcellular localization of TMEM33, the binding partners of TMEM33, and the expression of downstream effectors of PERK and IRE1?. Our data demonstrate that TMEM33 is a unique ER stress-inducible and ER transmembrane molecule, and a new binding partner of PERK. Exogenous expression of TMEM33 led to increased expression of p-eIF2? and p-IRE1? and their known downstream effectors, ATF4-CHOP and XBP1-S, respectively, in breast cancer cells. TMEM33 overexpression also correlated with increased expression of apoptotic signals including cleaved caspase-7 and cleaved PARP, and an autophagosome protein LC3II, and reduced expression of the autophagy marker p62. TMEM33 is a novel regulator of the PERK-eIE2?-ATF4 and IRE1-XBP1 axes of the UPR signaling. Therefore, TMEM33 may function as a determinant of the ER stress-responsive events in cancer cells.
Project description:Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces a highly conserved homeostatic response in all eukaryotic cells, termed the unfolded-protein response (UPR). Here we describe the characterization of stanniocalcin 2 (STC2), a mammalian homologue of a calcium- and phosphate-regulating hormone first identified in fish, as a novel target of the UPR. Expression of STC2 gene is rapidly upregulated in cultured cells after exposure to tunicamycin and thapsigargin, by ATF4 after activation of the ER-resident kinase PERK. In addition, STC2 expression is also activated in neuronal cells by oxidative stress and hypoxia but not by several cellular stresses unrelated to the UPR. In contrast, expression of another homologue, STC1, is only upregulated by hypoxia independent of PERK or ATF4 expression. In vivo studies revealed that rat cortical neurons rapidly upregulate STC2 after transient middle cerebral artery occlusion. Finally, siRNA-mediated inhibition of STC2 expression renders N2a neuroblastoma cells and HeLa cells significantly more vulnerable to apoptotic cell death after treatment with thapsigargin, and overexpression of STC2 attenuated thapsigargin-induced cell death. Consequently, induced STC2 expression is an essential feature of survival component of the UPR.
Project description: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.
Project description:HIF1? (hypoxia inducible factor 1?) is the central regulator of the cellular response to low oxygen and its activity is deregulated in multiple human pathologies. Consequently, given the importance of HIF signaling in disease, there is considerable interest in developing strategies to modulate HIF1? activity and down-stream signaling events. In the present study we find that under hypoxic conditions, activation of the PERK branch of the unfolded protein response (UPR) can suppress the levels and activity of HIF1? by preventing efficient HIF1? translation. Activation of PERK inhibits de novo HIF1? protein synthesis by preventing the RNA-binding protein, YB-1, from interacting with the HIF1? mRNA 5'UTR. Our data indicate that activation of the UPR can sensitise tumor cells to hypoxic stress, indicating that chemical activation of the UPR could be a strategy to target hypoxic malignant cancer cells.
Project description:Endoplasmic reticulum (ER) stress is thought to activate autophagy via unfolded protein response (UPR)-mediated transcriptional up-regulation of autophagy machinery components and modulation of microtubule-associated protein 1 light chain 3 (LC3). The upstream UPR constituents pancreatic EIF2-? kinase (PERK) and inositol-requiring enzyme 1 (IRE1) have been reported to mediate these effects, suggesting that UPR may stimulate autophagy via PERK and IRE1. However, how the UPR and its components affect autophagic activity has not been thoroughly examined. By analyzing the flux of LC3 through the autophagic pathway, as well as the sequestration and degradation of autophagic cargo, we here conclusively show that the classical ER stressor tunicamycin (TM) enhances autophagic activity in mammalian cells. PERK and its downstream factor, activating transcription factor 4 (ATF4), were crucial for this induction, but surprisingly, IRE1 constitutively suppressed autophagic activity. TM-induced autophagy required autophagy-related 13 (ATG13), Unc-51-like autophagy-activating kinases 1/2 (ULK1/ULK2), and GABA type A receptor-associated proteins (GABARAPs), but interestingly, LC3 proteins appeared to be redundant. Strikingly, ATF4 was activated independently of PERK in both LNCaP and HeLa cells, and our further examination revealed that ATF4 and PERK regulated autophagy through separate mechanisms. Specifically, whereas ATF4 controlled transcription and was essential for autophagosome formation, PERK acted in a transcription-independent manner and was required at a post-sequestration step in the autophagic pathway. In conclusion, our results indicate that TM-induced UPR activates functional autophagy, and whereas IRE1 is a negative regulator, PERK and ATF4 are required at distinct steps in the autophagic pathway.
Project description:XBP1 is a multitasking transcription factor and a key component of the unfolded protein response (UPR). Despite the wealth of knowledge about the role of XBP1 in luminal/ER-positive breast cancer, not much is known about the effectors of XBP1 in this context. Here we show that NCOA3 is a transcriptional target of XBP1. We observed increased expression of NCOA3 during conditions of UPR and oestrogen (E2) stimulation. Further investigations revealed a role for the IRE1-XBP1 axis in the induction of NCOA3 during UPR and oestrogen signalling. We identify a novel role for NCOA3 in activation of PERK-ATF4 axis during UPR where knockdown of NCOA3 compromised the optimal activation of the PERK-ATF4 pathway. We found that NCOA3 is required for induction of XBP1 during E2 stimulation and uncover a positive feedback regulatory loop that maintains high levels of NCOA3 and XBP1 in breast cancer. Furthermore, upregulated NCOA3 was required for XBP1-mediated resistance to antihormonal agents. Increased expression of NCOA3 was associated with poor prognosis and higher levels of XBP1-S in breast cancer tissues. Our results uncover a novel steroid hormone-independent role for NCOA3 in UPR signalling. Further we identify a positive feedback regulatory loop consisting of XBP1 and NCOA3 that maintains high levels of NCOA3 and XBP1 expression in breast cancer tissues. Taken together our data identify XBP1-NCOA3 axis that regulates cell fate decisions in ER-positive breast cancer cells.