Cell Death Discovery

Fas-associated death domain protein (FADD) plays a vital role in the extrinsic apoptotic pathway, where it forms an essential component of the death-inducing signaling complex (DISC). However, the precise early molecular events that facilitate recruitment of FADD to the DISC remain poorly defined. Using affinity purification and mass spectrometry we investigated the FADD interactome in untreated cells and following death receptor stimulation to identify novel FADD-interacting proteins. As expected, in death receptor-stimulated samples our analysis identified key components of the DISC such as Caspase-8. In addition, we identified novel binding partners including Transferrin Receptor 1 (TfR1) and Myosin Light Chain Kinase 2 (MYLK2) that are able to modulate FADD recruitment to the DISC and consequently downstream apoptotic signaling. TfR1 is pre-associated with FADD and recruited into the DISC; moreover, our data reveal that TfR1 is also pre-associated with the death receptors, TRAIL-R1 and TRAIL-R2, thereby functioning as a key regulator of DISC formation. In the case of MYLK2, specific binding of FADD to MYLK2 in non-apoptotic cells sequesters FADD from other DISC components ensuring aberrant apoptosis is not initiated. Furthermore, MYLK2 enzymatic activity is required to for it to translocate, in complex with FADD, to sites of DISC-mediated death receptor oligimerization. Taken together, our study highlights the important role that additional novel FADD binding partners play in the regulation of death receptor-mediated apoptotic cell death, in part by modulating FADD recruitment to the DISC.

Apoptosis inhibitor 5 (Api5) is an inhibitor of apoptosis, which is found to be upregulated in several cancers and promotes invasion as well as metastasis. Over-expression of Api5 is positively co-related with poor survival of cancers and inhibition of DNA damage induced apoptosis in cancerous cells. Acetylation at lysine 251 (K251) on Api5 facilitates the stability of the protein and thus functionally provides resistance to cancer cells against chemotherapeutic or anti-cancerous agents. However, the regulation of Api5 upon DNA damage is not yet known. In this study, we demonstrate that Api5 undergoes degradation following DNA damage via the ubiquitin-proteasome system. Upon DNA damage, ATR was observed to phosphorylate Api5 at serine 138 which led to the cytoplasmic localisation of Api5. The E3-ubiquitin ligase, SCF-FBXW2 ubiquitinates Api5 leading to its proteasomal degradation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 infection has led to worsened outcomes for patients with cancer. SARS-CoV-2 spike protein mediates host cell infection and cell-cell fusion that causes stabilization of tumor suppressor p53 protein. In-silico analysis previously suggested that SARS-CoV-2 spike interacts with p53 directly but this putative interaction has not been demonstrated in cells. We examined the interaction between SARS-CoV-2 spike, p53 and MDM2 (E3 ligase, which mediates p53 degradation) in cancer cells using an immunoprecipitation assay. We observed that SARS-CoV-2 spike protein interrupts p53-MDM2 protein interaction but did not detect SARS-CoV-2 spike bound with p53 protein in the cancer cells. We further observed that SARS-CoV-2 spike suppresses p53 transcriptional activity in cancer cells including after nutlin exposure of wild-type p53-, spike S2-expressing tumor cells and inhibits chemotherapy-induced p53 gene activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2. The suppressive effect of SARS-CoV-2 spike on p53-dependent gene activation provides a potential molecular mechanism by which SARS-CoV-2 infection may impact tumorigenesis, tumor progression and chemotherapy sensitivity. In fact, cisplatin-treated tumor cells expressing spike S2 were found to have increased cell viability as compared to control cells. Further observations on {gamma}-H2AX expression in spike S2-expressing cells treated with cisplatin may indicate altered DNA damage sensing in the DNA damage response pathway. The preliminary observations reported here warrant further studies to unravel the impact of SARS-CoV-2 and its various encoded proteins including spike on pathways of tumorigenesis and response to cancer therapeutics.

Human silencers have been shown to exist and regulate developmental gene expression. However, the functional importance of human silencers needs to be elucidated such as the working mechanism and whether they can form "super-silencers". Here, through interrogating two putative silencer components of FGF18 gene, we found that two silencers can cooperate via compensated chromatin interactions to form a "super-silencer". Furthermore, double knock-out of two silencers exhibited synergistic upregulation of FGF18 expression and changes of cell identity. To disturb the "super-silencers", we applied combinational treatment of an EZH2 inhibitor GSK343, and a REST inhibitor, X5050 ("GR"). We found that GR led to severe loss of TADs and loops, while the use of just one inhibitor by itself only showed mild changes. Such changes of TADs and loops may due to reduced CTCF protein level observed upon GR treatment. Moreover, GSK343 and X5050 worked together synergistically to upregulate the apoptotic genes controlled by super-silencers, and thus gave rise to antitumor effects including apoptosis, cell cycle arrest and tumor growth inhibition. Overall, our data demonstrated the first example of a "super-silencer" and showed that combinational usage of GSK343 and X5050 could potentially lead to cancer ablation through disruption of "super-silencers".

Understanding how the malignant cells respond to chemotherapy is essential to prevent the development of resistance and to improve the efficiency of anti-cancer drugs. Recently, we established that, by intrinsic and paracrine mechanisms, taxol treatment in breast tumor cells increases NOXA a pro-apoptotic protein functioning as an endogenous inhibitor of survival protein MCL-1, thereby enhancing cytotoxic load on the compensatory survival protein BCL-xL. We herein sought to define the contribution of NOXA/MCL-1 to the modality of cell death secretome composition upon anti-mitotic treatment associated with a BCL-xL antagonist. We observed that genetic inactivation of NOXA (enforcing MCL-1 pro-survival activity) in cancer cells not only delays their death when exposed to taxol in combination with the BCL-xL antagonist A1331852, but also alters its morphological characteristics with the apparition of features evoking pyroptosis. We identified the Caspase3-GSDME axis as regulating pyroptotic-like features suggesting that NOXA may act as a negative regulator of this cell death process (and MCL-1 as a positive regulator for it). Furthermore, comparative analysis of secretomes from the NOXA proficient or deficient cancer cells treated by taxol reveals variations in inflammatory cytokine production including those of IL-1{beta} and IL-18. Thus, our results show that anti-mitotic treatments are able to induce death by apoptosis and/or pyroptosis depending on BCL-2 family balance in breast cancer cells. Furthermore, NOXA/MCL-1 ratio appears to control the communication between these two types of cell death and their associated extracellular inflammatory signals in coordination with the pore-forming gasdermin GSDME.

Mitochondrial damage is a key feature of regulated cell death in neurons. In particular, mitochondrial outer membrane permeabilization (MOMP) has been proposed as a starting point for mitochondrial demise upon cellular stress. Potential mechanisms for MOMP presented in the literature include membrane pore formation by Bcl2-family proteins such as BID and BAX, oligomerization of voltage-dependent anion channels (VDACs) and hetero-oligomer formation of these proteins. In our study, we demonstrate a direct interaction between the voltage-dependent anion channel VDAC1 and the pro-apoptotic protein BID in dying neurons both in vitro and in vivo. Binding of BID to VDAC1 affects anion conductance through VDAC1 and is associated with glutamate-induced cell death in cultured neurons and ischemic brain injury. In cultured neurons, reducing VDAC1 expression significantly attenuates BID-induced hallmarks of mitochondrial damage such as mitochondrial fission, declined mitochondrial respiration, increased ROS production, and mitochondrial membrane potential breakdown. Our data highlight a critical role for VDAC1 as a mitochondrial receptor for activated BID, thereby serving as a key decision point between life and death in neurons. One Sentence SummaryVDAC1 interacts with BID to mediate mitochondrial membrane permeabilization and neuronal cell death.

We have previously identified sphingolipid metabolism as a key intracellular process associated with immunogenic cell death (ICD) induced by mitoxantrone in colon cancer cells. Specifically, we have demonstrated that inhibition of the sphingosine kinases (SphKs) synergistically enhanced production of hallmarks of ICD including ectoCRT production. To better understand the mechanism associated with ICD enhanced by SphK1-inhibition, we focused on the ER stress-associated intracellular signaling pathways leading to ectoCRT production. It is known that ABT-263 and AZD-5991 (ABT/AZD) are inhibitors of Bcl-2/Bcl-XL and MCL-1, respectively, leading to activation of Bak/Bax. Herein, we now provide evidence that treatment of DLD-1 colon cancer cells with ABT/AZD results in the production of ectoCRT indicative of ICD. Additionally, our data show that ABT/AZD-induced ectoCRT production is significantly enhanced by combination treatment with the SphK1 inhibitor, PF-543. Mechanistically, we demonstrate that combined treatment of ABT/AZD+PF-543 induces ectoCRT exposure in a caspase 8-dependent manner. Accordingly, we have identified a Bak/Bax activation-dependent pathway that leads to activation of a pro-survival SphK1/sphingosine-1-phosphate (S1P) signaling that attenuates ectoCRT production. Additionally, we have identified a regulatory role of ceramide synthase 6 (CerS6)- C16:0 Cer in transporting of dimeric CRT to the cell surface (ectoCRT). Together, these results indicate that sphingolipid metabolites, such as S1P and C16:0 Cer, have a key regulatory role for survival/death decisions of cancer cells in response to ICD-inducing chemotherapeutic agents such as mitoxantrone and ABT/AZD. Hence, targeting SphKs may be an innovative means to enhance the efficacy of ICD-inducing chemotherapeutic agents promoting anti-tumor innate/adaptive immune response since SphK inhibition blocks the anti-ICD effects of S1P while simultaneously accumulating sphingosine (Sph) leading to pro-ICD C16:0 Cer synthesis.

Superoxide dismutase 1 (SOD1) catalyzes the superoxide conversion to oxygen and hydrogen peroxide in general. SOD1 can translocate to the cell nucleus in response to oxidative stress in yeast and human fibroblasts. Here, we report the translocation of sod1 to the cell nucleus in primary co-cultures of neurons and astrocytes derived from newborn rats explants even in the absence of oxidative stress stimuli. The successful tissue explants from rats allowed simplistic and clean modeling of Amyotrophic lateral sclerosis (ALS)-simile tissue microenvironment. This mixed-cells-population was responsive to H2O2 (100 M) oxidative stress with a progressive dying process up to 6 h after stimuli. However, no differences in SOD1 nucleus translocation were observed in rat amyotrophic lateral sclerosis (ALS)-simile tissue microenvironment up to 6 hours under oxidative stress. These results altogether point to a robust cellular collaboration between neurons and astrocytes so that the tissue can equilibrate this SOD1 translocation during the oxidative stress response and in the early animal development.

The structure and function of the mitochondrial network are finely regulated. Among the proteins involved in these regulations, mitochondrial dynamics actors have been reported to regulate the apoptotic process. We show here in the Drosophila model that the mitophagy inducers, PINK1 (PTEN- induced putative kinase 1) and BNIP3 (Bcl-2 Interacting Protein 3), modulate mitochondrial apoptosis differently. If close links between the fission-inducing protein DRP1 and Bcl-2 family proteins, regulators of apoptosis, are demonstrated, the connection between mitophagy and apoptosis is still poorly understood. In Drosophila, we have shown that Rbf1, a homolog of the oncosuppressive protein pRb, induces cell death in proliferating larval tissues through a mechanism involving the interaction of Drp1 with Debcl, a pro-apoptotic protein of the Bcl-2 family. This interaction is necessary to induce mitochondrial fission, ROS production, and apoptosis. To better understand the interactions between the proteins involved in mitochondrial homeostasis and the apoptotic process, we focused on the role of two known players in mitophagy, the proteins PINK1 and BNIP3, during mitochondrial apoptosis induced by Rbf1 and Debcl in a proliferating Drosophila larval tissue. We show that Rbf1- or Debcl-induced apoptosis is accompanied by mitophagy. Interestingly, PINK1 and BNIP3 have distinct effects in regulating cell death. PINK1 promotes rbf1- or debcl-induced apoptosis, whereas BNIP3 protects against Rbf1-induced apoptosis but reduces Debcl- induced tissue loss without inhibiting Debcl-induced cell death. Furthermore, our results indicate that BNIP3 is required to induce basal mitophagy while PINK1 is responsible for mitophagy induced by rbf1 overexpression. These results highlight the critical role of mitophagy regulators in controlling homeostasis and cell fate.

The lysosomal damage response is important for the maintenance of cellular homeostasis. Although the mechanisms underlying the repair and autophagic elimination of damaged lysosomes have been elucidated, the early signal transduction pathways and genes induced in response to lysosomal damage remain elusive. We performed transcriptome and proteome analyses and found that the TAB-TAK1- IKK-NF-{kappa}B axis is activated by K63-linked ubiquitin chains that accumulate on damaged lysosomes. This activates the expression of various transcription factors and cytokines that promote anti-apoptosis and intercellular signaling. The findings highlight the crucial role of ubiquitin-regulated signal transduction and gene expression in cell survival and cell-cell communication in response to lysosomal damage. The results suggest that the ubiquitin system is not only involved in the removal of damaged lysosomes by lysophagy, but also functions in the activation of cellular signaling for cell survival.

Thioneins are cysteine-rich apoproteins that regulate divalent metal homeostasis by virtue of their metal-chelation properties resulting in the ligand-bound metallothionein state. Previous studies show transient upregulation of the metallothionein (MT) gene cluster as part of a complex transcriptional response to a class of histone demethylase tool compounds targeting human Fe2+ dependent ketoglutarate oxygenases KDM6A (UTX) and KDM6B (JmjD3). The prototypic bioactive KDM6 inhibitor GSK-J4 induces apoptotic cell death in multiple myeloma cells and corresponding transcriptomic profiles are dominated by metal and metabolic stress response signatures, also observed in primary human myeloma cells. Here we investigate the hypothesis that metal-chelation by GSK-J4 provides the means for transport and intracellular release of Zn2+ leading to a metallothionein transcriptomic response signature. Live cell imaging of myeloma cells shows transient increases in intracellular free Zn2+ concentrations when exposed to GSK-J4, consistent with a model of inhibitor-mediated metal transport. Comparisons of GSK-J4 and ZnSO4 treatments in the presence or absence of metal chelators show that both treatment conditions induce different transcription factor repertoires with an overlapping MTF1 transcriptional regulation responsible for metallothionein and metal ion transport regulation. The data provide a possible explanation for the observed metal response upon GSK-J4 inhibition however the relationship with the pro-apoptotic mechanism in myeloma cells requires further investigation.

Mitochondria, often referred to as the "powerhouses of the cell," are particularly crucial in cancer cells due to their high energy demands. Mitochondrial fusion-fission dynamics play a critical role in regulating signaling pathways and metabolic activities in colorectal cancer (CRC) cells. Increased mitochondrial fission drives metabolic reprogramming, enabling CRCs to proliferate, metastasize, and resist chemotherapy. Paradoxically, excessive fission induces mitochondria-mediated apoptosis. Our previous studies have shown that hematopoietic stem cell-derived conditioned media (CM) modulate the apoptosis pathway and mitochondrial bioenergetics of cancer stem cells by altering the cancer microenvironment. In this study, we found that HSCs-CM facilitates excessive fission in colorectal cancer cells by modulating Drp-1 and inducing the mitophagy-mediated apoptosis pathway, leading to the clearance of these cells. Moreover, proteomics data showed that HSCs-CM dysregulated the electron transport chain complexes, with an exceptionally high degree of dysregulation of complexes III and IV. Metabolomics and RNA sequencing revealed the dysregulation of critical proteins involved in mitochondrial bioenergetics and the autophagy pathway in CRCs treated with CM. Taken together, our studies reveal the therapeutic potential of HSC-conditioned media for treating colorectal cancer. HighlightsO_LIHematopoietic stem cell-derived conditioned media induced excessive mitochondrial fission by upregulating the Drp-1 protein, leading to the upregulation of the apoptosis pathway and cell death. C_LIO_LIThe excessive mitochondrial fission and bioenergetic dysfunction induced by HSCs-CM result in a loss of mitochondrial membrane potential (MMP) and high reactive oxygen species (ROS) production C_LIO_LIHSCs-CM severely disrupt mitochondrial bioenergetics in CRC cells, leading to an energy crisis and promoting the PINK-1 mediated mitophagy pathway. C_LI

AIF1 overexpression is intimately linked to the sensitivity of the yeast cells towards hydrogen peroxide or acetic acid. Therefore, studying the mechanism of its regulation in the cell would provide a significant understanding of the factors ultimately guiding yeast apoptosis. In this report, we establish the time-dependent induction of AIF1 in hydrogen peroxide stress. Additionally, the AIF1 expression in hydrogen peroxide is mediated by two transcription factors, Yap5 (DNA binding) and Cdc73 (non-DNA binding). Furthermore, substituting the H3K36 residue with another significantly abrogates the AIF1 expression. However, substituting H3K4 or H3K79 with A does not affect the AIF1 expression level under hydrogen peroxide stress. Altogether, the significant reduction of AIF1 expression in cdc73{Delta} cells plausibly reflects the reduced H3K36me3 modification and is independent of the H3K4me3 modification.

The activation of tumor necrosis factor receptors (TNFR) controls pleiotropic pro-inflammatory functions ranging from apoptosis to survival. The ability to trigger a particular function will depend on the upstream activation, association with regulatory complexes and downstream pathways. In Drosophila, two TNFRs have been identified, Wengen (Wgn) and Grindelwald (Grnd). Although several reports associate these receptors with JNK-dependent apoptosis, it has recently been found that Wgn activates a variety of functions. We demonstrate that Wgn is required for survival by protecting cells from apoptosis. This is mediated by the signaling molecule dTRAF1 and results in the activation of the p38 MAP kinase signaling pathway. Remarkably, Wgn is required for apoptosis-induced regeneration and is activated by the reactive oxygen species (ROS) produced following apoptosis. This ROS activation is exclusive for Wgn, but not for Grnd, and occurs in the absence of the ligand Eiger/TNF. Furthermore, based on protein sequence conservation, the extracellular Cys-rich domain of Grnd is much more divergent and phylogenetically restricted than that of Wgn, which is more similar to TNFR families from other animals, including those of human TNFRs. Taken together, our results show a novel function for a TNFR that responds to cellular damage by ensuring the cell survival required for the response to damage.

Mitochondrial dynamics are emerging as master regulators for targeting several types of cancers, including breast cancer, cervical cancer, and hepatocellular carcinoma, for therapeutic intervention. Mitochondrial morphology, size, position and activity within cells is regulated by dynamic fission and fusion events. Dynamin-related protein 1 (Drp1) promotes mitochondrial fission and maintains mitochondrial homeostasis. Loss of Scrib is implicated in several human cancers wherein mitochondrial dysfunction leads to excessive cell proliferation and metastasis. However, the exact molecular mechanisms behind the Scrib loss induced dysregulation of mitochondrial dynamics in cancer progression remains obscure. Although the role of mitochondrial dynamics are being investigated in several types of cancers, but the role of Drp1- mediated fission event in regulating the maintenance of polarity of cells upon loss of Scrib function is elusive. In this study, for the first time, we blocked the function of Drp1 activity in Scrib knockdown induced metastasis cancer model by two ways, firstly, through genetic ablation of Drp1, and secondly by using mdivi-1, a Drp1 specific inhibitor. Genetic depletion of Drp1 expression (Drp1RNAi) in Scrib knockdown cells inhibits Metalloproteinase MMP1, reduces ROS production, restores apico-basal (A/B) cell polarity and enhances ATP production. Further to confirm role of Drp1 in regulation of cell polarity, we employed mdivi, a Drp1 specific inhibitor which has dose dependent effect in cell polarity regulation. This study also reveals that JNK inhibition (JNKRNAi) in Scrib abrogated cells mitigates the Drp1 expression and controls cell proliferation leading to restoration of mitochondrial morphology and epithelial cellpolarity. Our results highlight Drp1 as a key regulator in maintaining the apico-basal polarity of cells which gets affected upon loss of Scrib but Drp1-JNK downregulation effectively mitigates ScribRNAi associated cell proliferation, metastasis and pupal lethality phenotypes.

Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down NAD+, it is also allosterically inhibited by NAD+. However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a "biological trigger" of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP1 inhibitor, PJ34 restored mitochondrial homeostasis and prevented subsequent Sarm1 activation in rotenone treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone mediated loss of locomotor abilities and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncovers a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders mediated by Sarm1.

During programmed cell death (PCD), it is commonly accepted that macrophages are recruited by apoptotic cells to complete cell degradation. Interdigital cell death, a classical model of PCD, contributes to digit individualization in limbs of mammals and other vertebrates. Here we show that macrophages are present in interdigits before significant cell death occurs and remain after apoptosis inhibition. The typical interdigital phagocytic activity was not observed after a partial depletion of macrophages and was markedly reduced by engulfment/phagosome maturation inhibition, as detected by its association with high lysosomal activity. {beta}-galactosidase activity in this region was also coupled with phagocytosis, against its relationship with cellular senescence. Interdigital phagocytosis correlated with high levels of reactive oxygen species (ROS), common in embryo regions carrying abundant cell death, suggesting that macrophages are the major source of ROS. ROS generation was dependent on NADPH oxidases and blood vessel integrity, but not directly associated with lysosomal activity. Therefore, macrophages prepattern regions where abundant cell death is going to occur, and high lysosomal activity and the generation of ROS by an oxidative burst-like phenomenon are activities of phagocytosis. Summary statementRecruitment of macrophages to the interdigital regions is not linked to apoptosis initiation and they phagocytize by a mechanism involving high lysosomal activity and an oxidative burst-like phenomenon.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB-mediated acetylation serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to extrinsic pro-apoptotic stimuli, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway impacts apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

An unmet challenge in managing breast cancer is treatment failure due to resistance to apoptosis-inducing chemotherapies. Thus, it is important to identify novel non-apoptotic therapeutic agents. Several non-apoptotic programmed cell death pathways utilize specific cellular signaling events to trigger lytic and pro-inflammatory cell death. PANoptosis, which encompasses pyroptosis, apoptosis and necroptosis, is of paramount importance in the regulation of cell death and immune responses. Our study illustrates that ophiobolin A (OpA) is an anti-cancer agent that triggers lytic cell death in breast cancer cells, including triple-negative breast cancer (TNBC), via a mechanism dependent on RIPK1. This study reveals that OpA induces typical pyroptosis-like characteristics, including cellular swelling, plasma membrane rupture, GSDMD cleavage and release of cytokines in breast cancer cells. The involvement of caspase 3, RIPK1, and GSDMD suggests that PANoptosis is activated upon OpA treatment in breast cancer. The induction of pro-inflammatory cell death suggests potential applications for OpA in cancer treatment.

Emerging evidence has highlighted the importance of targeting EZH2 in bladder cancer owing to the highly mutated nature of bladder cancers harboring mutations in chromatin regulatory genes opposing Polycomb-mediated repression. Besides, enhanced expression of EZH2 contributes to pathogenesis. Furthermore, the critical role of the retinoic acid signaling pathway in the development and homeostasis of the urothelium is well established. Here we report that coordinated targeting of EZH2 and the retinoic acid signaling pathway caused cytotoxic effects on bladder cancer cells by inducing a synergistic reduction in proliferative potential that was associated with increased apoptosis and cell cycle arrest in a cooperative and orchestrated manner. Moreover, combined treatment caused the modulation of the expression of genes associated with an anti-oncogenic profile, as reflected by the stimulation of marker genes associated with apoptosis and differentiation. We further portrayed a molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of certain genes associated with unfolded protein response and some metabolic processes. This work also characterized an apoptotic program centered on the master transcriptional regulators C/EBP{beta} and CHOP. These findings highlight the importance of co-targeting the EZH2/retinoic acid pathway in bladder cancers and encourage the design of novel treatments employing retinoids coupled with EZH2 inhibitors in bladder carcinoma.