Unconventional Ways to Live and Die: Cell Death and Survival in Development, Homeostasis, and Disease.

Balancing cell death and survival is essential for normal development and homeostasis and for preventing diseases, especially cancer. Conventional cell death pathways include apoptosis, a form of programmed cell death controlled by a well-defined biochemical pathway, and necrosis, the lysis of acutely injured cells. New types of regulated cell death include necroptosis, pyroptosis, ferroptosis, phagoptosis, and entosis. Autophagy can promote survival or can cause death. Newly described processes of anastasis and resuscitation show that, remarkably, cells can recover from the brink of apoptosis or necroptosis. Important new work shows that epithelia achieve homeostasis by extruding excess cells, which then die by anoikis due to loss of survival signals. This mechanically regulated process both maintains barrier function as cells die and matches rates of proliferation and death. In this review, we describe these unconventional ways in which cells have evolved to die or survive, as well as the contributions that these processes make to homeostasis and cancer.

Blebs promote cell survival by assembling oncogenic signalling hubs.

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

Loss of NR5A1 in mouse Sertoli cells after sex determination changes cellular identity and induces cell death by anoikis.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

Glutamate Dehydrogenase to the Rescue.

Tumor cell metabolism can be altered to support specific pathological functions or to adapt to environmental stresses. In this issue of Molecular Cell, Jin et al. (2018) identify induction of glutamate dehydrogenase as a critical metabolic adaptation in matrix-detached cancer cells that is required for metastasis of LKB1-deficient lung tumors.

The PLAG1-GDH1 Axis Promotes Anoikis Resistance and Tumor Metastasis through CamKK2-AMPK Signaling in LKB1-Deficient Lung Cancer.

Loss of LKB1 is associated with increased metastasis and poor prognosis in lung cancer, but the development of targeted agents is in its infancy. Here we report that a glutaminolytic enzyme, glutamate dehydrogenase 1 (GDH1), upregulated upon detachment via pleomorphic adenoma gene 1 (PLAG1), provides anti-anoikis and pro-metastatic signals in LKB1-deficient lung cancer. Mechanistically, the GDH1 product α-KG activates CamKK2 by enhancing its substrate AMPK binding, which contributes to energy production that confers anoikis resistance. The effect of GDH1 on AMPK is evident in LKB1-deficient lung cancer, where AMPK activation predominantly depends on CamKK2. Targeting GDH1 with R162 attenuated tumor metastasis in patient-derived xenograft model and correlation studies in lung cancer patients further validated the clinical relevance of our finding. Our study provides insight into the molecular mechanism by which GDH1-mediated metabolic reprogramming of glutaminolysis mediates lung cancer metastasis and offers a therapeutic strategy for patients with LKB1-deficient lung cancer.

Identification and immunological characteristics of anoikis-associated molecular clusters in lung adenocarcinoma.

Anoikis plays a crucial role in the progression, prognosis, and immune response of lung adenocarcinoma (LUAD). However, its specific impact on LUAD remains unclear. In this study, we investigated the intricate interplay of nesting apoptotic factors in LUAD. By analyzing nine key nesting apoptotic factors, we categorized LUAD patients into two distinct clusters. Further examination of immune cell profiles revealed that Cluster A exhibited greater infiltration of innate immune cells than did Cluster B. Additionally, we identified two genes closely associated with prognosis and developed a predictive model to differentiate patients based on molecular clusters. Our findings suggest that the loss of specific anoikis-related genes could significantly influence the prognosis, tumor microenvironment, and clinical features of LUAD patients. Furthermore, we validated the expression and functional roles of two pivotal prognostic genes, solute carrier family 2 member 1 (SLC2A1) and sphingosine kinase 1 (SPHK1), in regulating tumor cell viability, migration, apoptosis, and anoikis. These results offer valuable insights for future mechanistic investigations. In conclusion, this study provides new avenues for advancing our understanding of LUAD, improving prognostic assessments, and developing more effective immunotherapy strategies.

Targeting anoikis resistance as a strategy for cancer therapy.

Anoikis, known as matrix detachment-induced apoptosis or detachment-induced cell death, is crucial for tissue development and homeostasis. Cancer cells develop means to evade anoikis, e.g. anoikis resistance, thereby allowing for cells to survive under anchorage-independent conditions. Uncovering the mechanisms of anoikis resistance will provide details about cancer metastasis, and potential strategies against cancer cell dissemination and metastasis. Here, we summarize the principal elements and core molecular mechanisms of anoikis and anoikis resistance. We discuss the latest progress of how anoikis and anoikis resistance are regulated in cancers. Furthermore, we summarize emerging data on selective compounds and nanomedicines, explaining how inhibiting anoikis resistance can serve as a meaningful treatment modality against cancers. Finally, we discuss the key limitations of this therapeutic paradigm and possible strategies to overcome them. In this review, we suggest that pharmacological modulation of anoikis and anoikis resistance by bioactive compounds could surmount anoikis resistance, highlighting a promising therapeutic regimen that could be used to overcome anoikis resistance in cancers.

Vimentin-mediated buffering of internal integrin ß1 pool increases survival of cells from anoikis.

BACKGROUND: The intermediate filament protein vimentin is widely recognized as a molecular marker of epithelial-to-mesenchymal transition. Although vimentin expression is strongly associated with cancer metastatic potential, the exact role of vimentin in cancer metastasis and the underlying mechanism of its pro-metastatic functions remain unclear. RESULTS: This study revealed that vimentin can enhance integrin ß1 surface expression and induce integrin-dependent clustering of cells, shielding them against anoikis cell death. The increased integrin ß1 surface expression in suspended cells was caused by vimentin-mediated protection of the internal integrin ß1 pool against lysosomal degradation. Additionally, cell detachment was found to induce vimentin Ser38 phosphorylation, allowing the translocation of internal integrin ß1 to the plasma membrane. Furthermore, the use of an inhibitor of p21-activated kinase PAK1, one of the kinases responsible for vimentin Ser38 phosphorylation, significantly reduced cancer metastasis in animal models. CONCLUSIONS: These findings suggest that vimentin can act as an integrin buffer, storing internalized integrin ß1 and releasing it when needed. Overall, this study provides insights regarding the strong correlation between vimentin expression and cancer metastasis and a basis for blocking metastasis using this novel therapeutic mechanism.

Identification of important genes related to anoikis in acute myocardial infarction.

Acute myocardial infarction (AMI) increasingly precipitates severe heart failure, with diagnoses now extending to progressively younger demographics. The focus of this study was to pinpoint critical genes linked to both AMI and anoikis, thereby unveiling potential novel biomarkers for AMI detection and intervention. Differential analysis was performed to identify significant differences in expression, and gene functionality was explored. Weighted gene coexpression network analysis (WGCNA) was used to construct gene coexpression networks. Immunoinfiltration analysis quantified immune cell abundance. Protein-protein interaction (PPI) analysis identified the proteins that interact with theanoikis. MCODE identified key functional modules. Drug enrichment analysis identified relevant compounds explored in the DsigDB. Through WGCNA, 13 key genes associated with anoikis and differentially expressed genes were identified. GO and KEGG pathway enrichment revealed the regulation of apoptotic signalling pathways and negative regulation of anoikis. PPI network analysis was also conducted, and 10 hub genes, such as IL1B, ZAP70, LCK, FASLG, CD4, LRP1, CDH2, MERTK, APOE and VTN were identified. IL1B were correlated with macrophages, mast cells, neutrophils and Tcells in MI, and the most common predicted medications were roxithromycin, NSC267099 and alsterpaullone. This study identified key genes associated with AMI and anoikis, highlighting their role in immune infiltration, diagnosis and medication prediction. These findings provide valuable insights into potential biomarkers and therapeutic targets for AMI.

ITGBL1 promotes anoikis resistance and metastasis in human gastric cancer via the AKT/FBLN2 axis.

The resistance to anoikis plays a critical role in the metastatic progression of various types of malignancies, including gastric cancer (GC). Nevertheless, the precise mechanism behind anoikis resistance is not fully understood. Here, our primary focus was to examine the function and underlying molecular mechanism of Integrin beta-like 1 (ITGBL1) in the modulation of anoikis resistance and metastasis in GC. The findings of our investigation have demonstrated that the overexpression of ITGBL1 significantly augmented the resistance of GC cells to anoikis and promoted their metastatic potential, while knockdown of ITGBL1 had a suppressive effect on both cellular processes in vitro and in vivo. Mechanistically, we proved that ITGBL1 has a role in enhancing the resistance of GC cells to anoikis and promoting metastasis through the AKT/Fibulin-2 (FBLN2) axis. The inhibition of AKT/FBLN2 signalling was able to reverse the impact of ITGBL1 on the resistance of GC cells to anoikis and their metastatic capability. Moreover, the expression levels of ITGBL1 were found to be significantly elevated in the cancerous tissues of patients diagnosed with GC, and there was a strong correlation observed between high expression levels of ITGBL1 and worse prognosis among individuals diagnosed with GC. Significantly, it was revealed that within our cohort of GC patients, individuals exhibiting elevated ITGBL1 expression and diminished FBLN2 expression experienced the worst prognosis. In conclusion, the findings of our study indicate that ITGBL1 may serve as a possible modulator of resistance to anoikis and the metastatic process in GC.

Cell aggregation activates small GTPase Rac1 and induces CD44 cleavage by maintaining lipid raft integrity.

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM to metastasize, induced lipid raft disruption in single cells, but lipid raft integrity was maintained in aggregated cells. We further found that lipid raft integrity in aggregated cells was required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promoted CD44 cleavage and generated CD44 intracellular domain (CD44 ICD) to enhance stemness of aggregated cells. Consequently, lipid raft disruption inhibited Rac1 activation, CD44 ICD generation, and metastasis. Our findings reveal two new pathways regulated by CD44-mediated cell aggregation via maintaining lipid raft integrity. These findings also suggest that targeting cell aggregation-mediated pathways could be a novel therapeutic strategy to prevent CTC cluster-initiated metastasis.

Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer.

Bladder cancer ranks as the 10th most common cancer worldwide, with deteriorating prognosis as the disease advances. While immune checkpoint inhibitors (ICIs) have shown promise in clinical therapy in both operable and advanced bladder cancer, identifying patients who will respond is challenging. Anoikis, a specialized form of cell death that occurs when cells detach from the extracellular matrix, is closely linked to tumor progression. Here, we aimed to explore the anoikis-based biomarkers for bladder cancer prognosis and immunotherapeutic decisions. Through consensus clustering, we categorized patients from the TCGA-BLCA cohort into two clusters based on anoikis-related genes (ARGs). Significant differences in survival outcome, clinical features, tumor immune environment (TIME), and potential ICIs response were observed between clusters. We then formulated a four-gene signature, termed "Ascore", to encapsulate this gene expression pattern. The Ascore was found to be closely associated with survival outcome and served as an independent prognosticator in both the TCGA-BLCA cohort and the IMvigor210 cohort. It also demonstrated superior predictive capacity (AUC = 0.717) for bladder cancer immunotherapy response compared to biomarkers like TMB and PD-L1. Finally, we evaluated Ascore's independent prognostic performance as a non-invasive biomarker in our clinical cohort (Gulou-Cohort1) using circulating tumor cells detection, achieving an AUC of 0.803. Another clinical cohort (Gulou-Cohort2) consisted of 40 patients undergoing neoadjuvant anti-PD-1 treatment was also examined. Immunohistochemistry of Ascore in these patients revealed its correlation with the pathological response to bladder cancer immunotherapy (P = 0.004). Impressively, Ascore (AUC = 0.913) surpassed PD-L1 (AUC = 0.662) in forecasting immunotherapy response and indicated better net benefit. In conclusion, our study introduces Ascore as a novel, robust prognostic biomarker for bladder cancer, offering a new tool for enhancing immunotherapy decisions and contributing to the tailored treatment approaches in this field.

A bibliometric and knowledge-map analysis of anoikis from 2003 to 2022.

This analysis covers 4494 anoikis-related publications (2003-2022). It explores annual trends, top countries, core journals, leading institutions, keywords, references, authors, and collaborations. Key findings include the United States leading in publications, Chulalongkorn University as the top institution, and Oncogene as the most prolific journal. The Journal of Biological Chemistry holds the highest influence. Burst keywords like "signal transduction," "apoptosis resistance," "metabolism," and "tumor microenvironment" highlight emerging research areas. This study offers a comprehensive overview, aiding researchers in grasping anoikis research trends, contributors, and prospects.

Circulating Tumor Cells: Does Ion Transport Contribute to Intravascular Survival, Adhesion, Extravasation, and Metastatic Organotropism?

Survival in the circulation, extravasation from vasculature, and colonizing new tissues represent major steps of the metastatic cascade and pose a big challenge for metastasizing tumor cells. Tumor cells circulating in blood and lymph vessels need to overcome anoikis, cope with mechanical stimuli including shear stress, and defeat attacks by the immune system. Once adhered to the vessel wall, a circulating tumor cell (CTC) can trick the endothelial cells into loosening their intercellular junctions so that the endothelium becomes penetrable for the tumor cell. Since tumor cells tend to metastasize to predestinated target organs and tissues, called organotropism, the distribution of metastases is anything but random. The molecular-physiological mechanisms underlying CTC survival, extravasation, and organotropism are very likely to include the presence and activity of ion channels/transporters due to the latter's key function in cytophysiological processes. To date, a very limited number of studies explicitly show the involvement of ion transport. This review describes the contribution of ion channels and transporters to CTC survival, extravasation, and organotropism where known and possible. In addition, supposed connections between ion transport and CTC behavior are demonstrated and imply the potential to be therapeutically taken advantage of.

TGFBR3 supports anoikis through suppressing ATF4 signaling.

Epithelial morphogenesis and oncogenic transformation can cause loss of cell adhesion, and detached cells are eliminated by anoikis. Here, we reveal that transforming growth factor ß receptor 3 (TGFBR3) acts as an anoikis mediator through the coordination of activating transcription factor 4 (ATF4). In breast cancer tissues, TGFBR3 is progressively lost, but elevated TGFBR3 is associated with a histologic subtype characterized by cellular adhesion defects. Dissecting the impact of extracellular matrix (ECM) deprivation, we demonstrate that ECM loss promotes TGFBR3 expression, which in turn causes differentiation of cell aggregates, conferring a low-adhesion phenotype, and drives the intrinsic apoptotic pathway. We demonstrate that inhibition of TGFBR3 impairs epithelial anoikis by activating ATF4 signaling. These preclinical findings provide a rationale for therapeutic inhibition of ATF4 in the subgroup of breast cancer patients with low TGFBR3 expression.

Unveiling Anoikis-related genes: A breakthrough in the prognosis of bladder cancer.

BACKGROUND: Bladder cancer (BLCA) is a prevalent malignancy worldwide. Anoikis remains a new form of cell death. It is necessary to explore Anoikis-related genes in the prognosis of BLCA. METHODS: We obtained RNA expression profiles from the The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases for dimensionality reduction analysis and isolated epithelial cells, T cells and fibroblasts for copy number variation analysis, pseudotime analysis and transcription factor analysis based on R package. We integrated machine-learning algorithms to develop the artificial intelligence-derived prognostic signature (AIDPS). RESULTS: The performance of AIDPS with clinical indicators was stable and robust in predicting BLCA and showed better performance in every validation dataset compared to other models. Mendelian randomization analysis was conducted. Single nucleotide polymorphism (SNP) sites of rs3100578 (HK2) and rs66467677 (HSP90B1) exhibited significant correlation of bladder problem (not cancer) and bladder cancer, whereasSNP sites of rs3100578 (HK2) and rs947939 (BAD) had correlation between bladder stone and bladder cancer. The immune infiltration analysis of the TCGA-BLCA cohort was calculated via the ESTIMATE (i.e. Estimation of STromal and Immune cells in MAlignantTumours using Expression data) algorithm which contains stromal, immune and estimate scores. We also found significant differences in the IC50 values of Bortezomib_1191, Docetaxel_1007, Staurosporine_1034 and Rapamycin_1084 among the high- and low-risk groups. CONCLUSIONS: In conclusion, these findings indicated Anoikis-related prognostic genes in BLCA and constructed an innovative machine-learning model of AIDPS with high prognostic value for BLCA.

A novel anoikis-related prognostic signature associated with prognosis and immune infiltration landscape in lung adenocarcinoma.

BACKGROUND: One of the most prevalent malignancies in the world is lung adenocarcinoma (LUAD), with a large number of people dying from lung cancer each year. Anoikis has a crucial function in tumor metastasis, promoting cancer cell shedding and survival from the primary tumor site. However, the role of anoikis in LUAD is still unclear. METHODS: The GeneCard database (https://www.genecards.org/) was utilized to obtain anoikis-related genes with correlation greater than 0.4. Differential analysis was employed to acquire differential genes. Univariate, multifactorial Cox analyses and the least absolute shrinkage and selection operator were then utilized to capture genes connected to overall survival time. These genes were used to build prognostic models. The predictive model was analyzed and visualized. Survival analysis was conducted on the model and risk scores were calculated. The TCGA samples were split into groups of low and high risk depending on risk scores. A Gene Expression Omnibus database sample was used for external verification. Immunization estimates were performed using ESTIMATE, CiberSort and single sample gene set enrichment analysis. The connection between the prognostic gene model and immune cells was analyzed. Drug susceptibility prediction analysis was performed. The clinical information for samples was extracted and analyzed. RESULTS: We selected six genes related to anoikis in LUAD to construct a prognosis model (CDC25C, ITPRIP, SLCO1B3, CDX2, CSPG4 and PIK3CG). Compared with cases of high-risk scores, the overall survival of those with low risk was significantly elevated based on Kaplan-Meier survival analysis. Immune function analysis exhibited that different risk groups had different immune states. The results of ESTIMATE, CiberSort and single sample gene set enrichment analysis showed great gaps in immunization between patients in the two groups. The normogram of the risk score and the LUAD clinicopathological features was constructed. Principal component analysis showed that this model could effectively distinguish the two groups of LUAD patients. CONCLUSIONS: We integrated multiple anoikis-related genes to build a prognostic model. This investigation demonstrates that anoikis-related genes can be used as a stratification element for fine therapy of individuals with LUAD.

Identification of prognostic value of anoikis-related gene score model combined with tumor microenvironment score models in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) has poor survival. Effective prognostic models with high application value remain lack. METHODS: Bulk RNA seq and single cell RNA-seq data were retrieved from the XENA-TCGA-ESCC cohort and GSE188900. The anoikis-related gene score (ANO score) model and tumor microenvironment score (TME score) model were constructed and merged into three subgroups. Functional annotation was analyzed by Gene Ontology terms. Univariate and multivariate Cox regression analysis, least absolute shrinkage and selection operator regression analysis and weighted gene coexpression network analysis were performed to construct prognostic prediction models and identify prognostic value. Kaplan-Meier survival curves were drawn for evaluating the overall survival (OS) of patients classified by different score subgroups. Immunotherapy response and mutation analyses were also conducted. RESULTS: In the ANO score model, TNFSF10 was an independent factor for the prognosis of ESCC patients. The area under the curve values of the ANO-TME score model in predicting the OS were 0.638 at 5 years and 0.632 at 7 years. Patients in the ANO low score-TME high score group had a much longer OS than patients in any other ANO-TME score subgroup (p < 0.001), suggesting a higher prognostic value. The differentially expressed genes of the ANO low score-TME high score group were mainly involved in cell adhesion molecules, nucleotide excision repair, the TGF-ß signaling pathway and mismatch repair. TP53 (92%), TTN (38%) and NFE2L2 (31%) were the top genes with highest mutant frequency in the ANO low score-TME high score group. CONCLUSIONS: A novel prognostic prediction model with high application value was constructed and identified for ESCC patients, which may provide evidence for immunotherapy in the treatment of ESCC.

Identification and validation of anoikis-related genes to clarify the prognosis and immune mechanisms of patients with low-grade glioma.

BACKGROUND: Low-grade glioma (LGG) has an extremely poor prognosis, and the mechanism leading to malignant development has not been determined. The aim of our study was to clarify the function and mechanism of anoikis and TIMP1 in the malignant progression of LGG. METHODS: We screened 7 anoikis-related genes from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to construct a prognostic-predicting model. The study assessed the clinical prognosis, pathological characteristics, and immune cell infiltration in both high- and low-risk groups. Additionally, the potential modulatory effects of TIMP1 on proliferation, migration, and anoikis in LGG were investigated both in vivo and in vitro. RESULTS: In this study, we identified seven critical genes, namely, PTGS2, CCND1, TIMP1, PDK4, LGALS3, CDKN1A, and CDKN2A. Kaplan‒Meier (K‒M) curves demonstrated a significant correlation between clinical features and overall survival (OS), and single-cell analysis and mutation examination emphasized the heterogeneity and pivotal role of hub gene expression imbalances in LGG development. Immune cell infiltration and microenvironment analysis further elucidated the relationships between key genes and immune cells. In addition, TIMP1 promoted the malignant progression of LGG in both in vitro and in vivo models. CONCLUSIONS: This study confirmed that TIMP1 promoted the malignant progression of LGG by inhibiting anoikis, providing insights into LGG pathogenesis and potential therapeutic targets.