Exploring the Communication of the SASP: Dynamic, Interactive, and Adaptive Effects on the Microenvironment.

Cellular senescence is a complex cell state that can occur during physiological ageing or after exposure to stress signals, regardless of age. It is a dynamic process that continuously evolves in a context-dependent manner. Senescent cells interact with their microenvironment by producing a heterogenous and plastic secretome referred to as the senescence-associated secretory phenotype (SASP). Hence, understanding the cross-talk between SASP and the microenvironment can be challenging due to the complexity of signal exchanges. In this review, we first aim to update the definition of senescence and its associated biomarkers from its discovery to the present day. We detail the regulatory mechanisms involved in the expression of SASP at multiple levels and develop how SASP can orchestrate microenvironment modifications, by focusing on extracellular matrix modifications, neighboring cells' fate, and intercellular communications. We present hypotheses on how these microenvironmental events may affect dynamic changes in SASP composition in return. Finally, we discuss the various existing approaches to targeting SASP and clarify what is currently known about the biological effects of these modified SASPs on the cellular environment.

Senescence Induced by UVB in Keratinocytes Impairs Amino Acids Balance.

Skin is one of the most exposed organs to external stress. Namely, UV rays are the most harmful stress that could induce important damage leading to skin aging and cancers. At the cellular level, senescence is observed in several skin cell types and contributes to skin aging. However, the origin of skin senescent cells is still unclear but is probably related to exposure to stresses. In this work, we developed an in vitro model of UVB-induced premature senescence in normal human epidermal keratinocytes. UVB-induced senescent keratinocytes display a common senescent phenotype resulting in an irreversible cell cycle arrest, an increase in the proportion of senescence-associated ß-galactosidase‒positive cells, unrepaired DNA damage, and a long-term DNA damage response activation. Moreover, UVB-induced senescent keratinocytes secrete senescence-associated secretory phenotype factors that influence cutaneous squamous cell carcinoma cell migration. Finally, a global transcriptomic study highlighted that senescent keratinocytes present a decrease in the expression of several amino acid transporters, which is associated with reduced intracellular levels of glycine, alanine, and leucine. Interestingly, the chemical inhibition of the glycine transporter SLC6A9/Glyt1 triggers senescence features.

The out-of-field dose in radiation therapy induces delayed tumorigenesis by senescence evasion.

A rare but severe complication of curative-intent radiation therapy is the induction of second primary cancers. These cancers preferentially develop not inside the planning target volume (PTV) but around, over several centimeters, after a latency period of 1-40 years. We show here that normal human or mouse dermal fibroblasts submitted to the out-of-field dose scattering at the margin of a PTV receiving a mimicked patient's treatment do not die but enter in a long-lived senescent state resulting from the accumulation of unrepaired DNA single-strand breaks, in the almost absence of double-strand breaks. Importantly, a few of these senescent cells systematically and spontaneously escape from the cell cycle arrest after a while to generate daughter cells harboring mutations and invasive capacities. These findings highlight single-strand break-induced senescence as the mechanism of second primary cancer initiation, with clinically relevant spatiotemporal specificities. Senescence being pharmacologically targetable, they open the avenue for second primary cancer prevention.

Cellular senescence and tumor promotion: Role of the Unfolded Protein Response.

Senescence is a cellular state which can be viewed as a stress response phenotype implicated in various physiological and pathological processes, including cancer. Therefore, it is of fundamental importance to understand why and how a cell acquires and maintains a senescent phenotype. Direct evidence has pointed to the homeostasis of the endoplasmic reticulum whose control appears strikingly affected during senescence. The endoplasmic reticulum is one of the sensing organelles that transduce signals between different pathways in order to adapt a functional proteome upon intrinsic or extrinsic challenges. One of these signaling pathways is the Unfolded Protein Response (UPR), which has been shown to be activated during senescence. Its exact contribution to senescence onset, maintenance, and escape, however, is still poorly understood. In this article, we review the mechanisms through which the UPR contributes to the appearance and maintenance of characteristic senescent features. We also discuss whether the perturbation of the endoplasmic reticulum proteostasis or accumulation of misfolded proteins could be possible causes of senescence, and-as a consequence-to what extent the UPR components could be considered as therapeutic targets allowing for the elimination of senescent cells or altering their secretome to prevent neoplastic transformation.

Unfolded Protein Response (UPR) Controls Major Senescence Hallmarks.

Senescence is a complex cellular state, which can be considered as a stress response phenotype. However, the mechanisms through which cells acquire and maintain this phenotype are not fully understood. In this paper, it is argued that the unfolded protein response (UPR) may represent a signalling platform that is associated with the major senescence hallmarks.

Connecting cancer relapse with senescence.

Many cancers respond to initial treatment but most of them relapse due to the persistence of dormant tumor cells. Determining the exact nature of the dormant state is crucial to develop therapies aiming to eradicate the dormant cells. Here, we argue that therapy-induced senescence of cancer cells could be an alternative form of dormancy.

Impact and Relevance of the Unfolded Protein Response in HNSCC.

Head and neck squamous cell carcinomas (HNSCC) encompass a heterogeneous group of solid tumors that arise from the upper aerodigestive tract. The tumor cells face multiple challenges including an acute demand of protein synthesis often driven by oncogene activation, limited nutrient and oxygen supply and exposure to chemo/radiotherapy, which forces them to develop adaptive mechanisms such as the Unfolded Protein Response (UPR). It is now well documented that the UPR, a homeostatic mechanism, is induced at different stages of cancer progression in response to intrinsic (oncogenic activation) or extrinsic (microenvironment) perturbations. This review will discuss the role of the UPR in HNSCC as well as in the key processes that characterize the physiology of HNSCC. The role of the UPR in the clinical context of HNSCC will also be addressed.

Alterations of EDEM1 functions enhance ATF6 pro-survival signaling.

Activating transcription factor 6 alpha (referred to as ATF6 hereafter) is an endoplasmic reticulum (ER)-resident glycoprotein and one of the three sensors of the unfolded protein response (UPR). Upon ER stress, ATF6 is exported to the Golgi complex where it is cleaved by the S1P and S2P proteases thus releasing ATF6 cytosolic fragment and leading to the transcription of ATF6 target genes. In this study, we performed a phenotypic small-interfering RNA (siRNA) screening to better characterize the ER mechanisms involved in ATF6 activation upon ER stress. This revealed that silencing of ER-degradation-enhancing alpha-mannosidase-like protein-1 (EDEM1) increased the bioavailability of ER stress-induced ATF6 export to the Golgi complex through the stabilization of the natively unstable ATF6 protein. Moreover, we characterized a somatic variant of EDEM1 (N198I) found in hepatocellular carcinoma that alters ATF6 signaling and might provide a selective advantage to the transforming cells. Hence, our work confirms the natively unstable nature of ATF6 and links this property to potentially associated pro-oncogenic functions.

Pre-malignant transformation by senescence evasion is prevented by the PERK and ATF6alpha branches of the Unfolded Protein Response.

The incidence of carcinomas highly increases with age. However, the initial steps of the age-related molecular carcinogenic processes remain poorly characterized. We previously showed that normal human epidermal keratinocytes spontaneously and systematically escape from senescence to give rise to preneoplastic emerging cells through a process called post-senescence neoplastic emergence (PSNE). To identify molecular pathways involved in the switch from senescence to pre-transformation, we performed Connectivity Map analyses and DAVID functional annotations followed by hierarchical clustering and multidimensional scaling of the gene expression signature of PSNE cells. We identified endoplasmic reticulum stress related pathways as key regulators of PSNE. Invalidation by RNA interference of the UPR sensors PERK, ATF6α, but not IRE1α, delayed the occurrence of senescence when performed in pre-senescent cells, and increased the PSNE frequency when performed in already senescent cells. Conversely, endoplasmic reticulum stress inducers applied to already senescent cells decreased the frequency of PSNE. In conclusion, these results indicate that the activation of the UPR could protect from the early carcinogenic steps by senescence evasion. This opens new avenues to explore therapeutics that could be useful in decreasing the age-associated tumor incidence.

Protein biosynthesis, a target of sorafenib, interferes with the unfolded protein response (UPR) and ferroptosis in hepatocellular carcinoma cells.

Sorafenib is the first line treatment for advanced hepatocellular carcinoma (HCC). We explored its impact on the proteostasis of cancer cells, i.e. the processes that regulate the synthesis, maturation and turn-over of cellular proteins. We observed that sorafenib inhibits the production of the tumour marker alpha-foetoprotein (AFP) in two different HCC cell lines, an effect that correlated with a radical inhibition of protein biosynthesis. This effect was observed at clinically relevant concentrations of sorafenib and was not related to the effect of sorafenib on the transport of amino acids across the plasma membrane or the induction of the unfolded protein response (UPR). Instead, we observed that sorafenib inhibits translation initiation and the mechanistic target of rapamycin (mTOR) signaling cascade, as shown by the analysis of phosphorylation levels of the protein 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1). We explored the consequences of this inhibition in HCC cells. We observed that overall sorafenib is a weak inducer of the UPR that can paradoxically prevent the UPR induced by tunicamycin. We also found no direct synergistic anticancer effect between sorafenib and various strategies that inhibit the UPR. In agreement with the possibility that translation inhibition might be an adaptive stress response in HCC cells, we noted that it protects cancer cell from ferroptosis, a form of oxidative necrosis. Our findings point to the modulation of protein biosynthesis and mTOR signaling as being important, yet complex determinants of the response of HCC cells to sorafenib.

Dual IRE1 RNase functions dictate glioblastoma development.

Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor aggressiveness. Evidence suggests that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This concept is valid in glioblastoma multiform (GBM), the most lethal primary brain cancer with no effective treatment. We previously demonstrated that the ER stress sensor IRE1α (referred to as IRE1) contributes to GBM progression, through XBP1 mRNA splicing and regulated IRE1-dependent decay (RIDD) of RNA Here, we first demonstrated IRE1 signaling significance to human GBM and defined specific IRE1-dependent gene expression signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes, mainly through selective remodeling of the tumor stroma. This study provides the first demonstration of a dual role of IRE1 downstream signaling in cancer and opens a new therapeutic window to abrogate tumor progression.

The ATF6α arm of the Unfolded Protein Response mediates replicative senescence in human fibroblasts through a COX2/prostaglandin E2 intracrine pathway.

Senescence is recognized as a cellular state acquired in response to various stresses. It occurs in correlation with the activation of the Unfolded Protein Response (UPR) pathway. However, the UPR targets which might relay the establishment of the senescent phenotype are not known. Herein, we investigated whether the up-regulation of the COX2 (PTGS2) limiting enzyme in the prostaglandin biosynthesis pathway, known to mediate cellular senescence in normal human fibroblasts, could be controlled by the UPR sensors ATF6α, IRE1α and PERK. We found that UPR inducers cause premature senescence through an increase in COX2 expression, and an overproduction of prostaglandin E2 (PGE2) in wild type fibroblasts but not in ATF6α invalidated ones. In replicative senescent fibroblasts, ATF6α and IRE1α silencing abrogated COX2 up-regulation and PGE2 production. The expanded ER and the large cell size characteristics of senescent fibroblasts were both reduced upon the invalidation of COX2 as well as ATF6α. These effects of the ATF6α invalidation were prevented by favoring the import of PGE2, but not just by supplying extracellular PGE2. Taken together, our results support a critical role of ATF6α in the establishment and maintenance of cellular senescence in normal human fibroblasts via the up-regulation of a COX2/PGE2 intracrine pathway.

Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?

Senescence is a cell state occurring in vitro and in vivo after successive replication cycles and/or upon exposition to various stressors. It is characterized by a strong cell cycle arrest associated with several molecular, metabolic and morphologic changes. The accumulation of senescent cells in tissues and organs with time plays a role in organismal aging and in several age-associated disorders and pathologies. Moreover, several therapeutic interventions are able to prematurely induce senescence. It is, therefore, tremendously important to characterize in-depth, the mechanisms by which senescence is induced, as well as the precise properties of senescent cells. For historical reasons, senescence is often studied with fibroblast models. Other cell types, however, much more relevant regarding the structure and function of vital organs and/or regarding pathologies, are regrettably often neglected. In this article, we will clarify what is known on senescence of epithelial cells and highlight what distinguishes it from, and what makes it like, replicative senescence of fibroblasts taken as a standard.

A potential role of the unfolded protein response in post-transplant cancer.

Cancer is one of the major causes of mortality in organ transplant patients receiving immunosuppressive regimen based on Cyclosporin A (CsA). Organ transplantation and chronic immunosuppression are typically associated with skin cancers (both squamous cell carcinoma and melanoma) and renal cell carcinoma (RCC). Recent studies have shown that in addition to its immunosuppressive effects, accounted for by the inhibition of calcineurin and the modulation of the transcriptional programme of lymphocytes, CsA also directly stimulates the growth and aggressive behaviour of various cancer cells. Using renal carcinogenesis as an example, we discuss the current evidence for a role of cellular proteostasis, i.e. the regulation of the production, maturation and turnover of proteins in eukaryotic cells, in tumorigenesis arising under conditions of chronic immunosuppression. We present the recent studies showing that CsA induces the unfolded protein response (UPR) in normal and transformed kidney cells. We examine how the UPR might be important, considering in particular the genomic analyses showing the existence of a correlation between the levels of expression of the actors of the UPR, the chaperones of the endoplasmic reticulum (ER) and the aggressiveness of renal carcinoma. The UPR may offer a possible explanation for how immunosuppressive regimens based on CsA promote renal carcinogenesis. We discuss the opportunities offered by this biological knowledge in terms of screening, diagnosis and treatment of post-transplant cancers, and propose possible future translational studies examining the role of tumour proteostasis and the UPR in this context.

Role of the unfolded protein response in tumor cell characteristics and cancer outcome.

PURPOSE OF REVIEW: In the present review, we discuss the possible role of the unfolded protein response (UPR) in the acquisition of tumor cell characteristics and in the prognosis of cancer outcome, which could assist and contribute to the development of more promising therapeutic strategies. RECENT FINDINGS: Accumulating evidence supports the idea that alteration of endoplasmic reticulum proteostasis is a key player in cancer development and aggressiveness. Some UPR components were reported as independent prognostic biomarker. Recent evidence supports a relationship between the UPR activation status and prognosis of tumors. This may represent an interesting avenue for better characterization of carcinogenesis and tumor type. SUMMARY: The contribution of the UPR to the characteristics of malignant tumors is complex and dependent on both intrinsic (e.g. oncogene addiction) and extrinsic (e.g. hypoxia) contexts. Through adaptation to severe microenvironmental conditions, UPR branches are generally a survival strategy for cancer cells, which are able to cope with this challenging context. We address the question of whether the activation status of the UPR is related to tumor properties and discuss the role of the UPR in the clinical context.

ATF6α regulates morphological changes associated with senescence in human fibroblasts.

Cellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated ß-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6α signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6α in senescent cells. Moreover, ATF6α drives the increase of senescence associated-ß-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6α in the establishment of morphological features of senescence in normal human primary fibroblasts.

Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells.

The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.

Alpha-fetoprotein is a biomarker of unfolded protein response and altered proteostasis in hepatocellular carcinoma cells exposed to sorafenib.

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting.