Transcriptome analysis of polyploid giant cancer cells and their progeny reveals a functional role for p21 in polyploidization and depolyploidization.

Polyploid giant cancer cells (PGCC) are frequently detected in tumors and are increasingly recognized for their roles in chromosomal instability and associated genome evolution that leads to cancer recurrence. We previously reported that therapy stress promotes polyploidy, and that acid ceramidase plays a role in depolyploidization. In this study, we used an RNA-seq approach to gain a better understanding of the underlying transcriptomic changes that occur as cancer cells progress through polyploidization and depolyploidization. Our results revealed gene signatures that are associated with disease-free and/or overall survival in several cancers and identified the cell cycle inhibitor CDKN1A/p21 as the major hub in PGCC and early progeny. Increased expression of p21 in PGCC was limited to the cytoplasm. We previously demonstrated that the sphingolipid enzyme acid ceramidase is dispensable for polyploidization upon therapy stress but plays a crucial role in depolyploidization. The current study demonstrates that treatment of cells with ceramide is not sufficient for p53-independent induction of p21 and that knockdown of acid ceramidase, which hydrolyzes ceramide, does not interfere with upregulation of p21. In contrast, blocking the expression of p21 with UC2288 prevented the induction of acid ceramidase and inhibited both the formation of PGCC from parental cells as well as the generation of progeny from PGCC. Taken together, our data suggest that p21 functions upstream of acid ceramidase and plays an important role in polyploidization and depolyploidization.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. This study reveals phenotypic consequences of whole-animal tetraploidy that make C. elegans an excellent model for ploidy differences.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

Crosstalk between pro-survival sphingolipid metabolism and complement signaling induces inflammasome-mediated tumor metastasis.

Crosstalk between metabolic and signaling events that induce tumor metastasis remains elusive. Here, we determine how oncogenic sphingosine 1-phosphate (S1P) metabolism induces intracellular C3 complement activation to enhance migration/metastasis. We demonstrate that increased S1P metabolism activates C3 complement processing through S1P receptor 1 (S1PR1). S1P/S1PR1-activated intracellular C3b-α'2 is associated with PPIL1 through glutamic acid 156 (E156) and aspartic acid 111 (D111) residues, resulting in NLRP3/inflammasome induction. Inactivation mutations of S1PR1 to prevent S1P signaling or mutations of C3b-α'2 to prevent its association with PPIL1 attenuate inflammasome activation and reduce lung colonization/metastasis in mice. Also, activation of the S1PR1/C3/PPIL1/NLRP3 axis is highly associated with human metastatic melanoma tissues and patient-derived xenografts. Moreover, targeting S1PR1/C3/PPIL1/NLRP3 signaling using molecular, genetic, and pharmacologic tools prevents lung colonization/metastasis of various murine cancer cell lines using WT and C3a-receptor1 knockout (C3aR1-/-) mice. These data provide strategies for treating high-grade/metastatic tumors by targeting the S1PR1/C3/inflammasome axis.

Polyploid giant cancer cells are dependent on cholesterol for progeny formation through amitotic division.

Polyploid Giant Cancer Cells (PGCC) are increasingly being recognized as drivers of cancer recurrence. Therapy stress promotes the formation of these cells, which upon stress cessation often successfully generate more aggressive progeny that repopulate the tumor. Therefore, identification of potential PGCC vulnerabilities is key to preventing therapy failure. We have previously demonstrated that PGCC progeny formation depends on the lysosomal enzyme acid ceramidase (ASAH1). In this study, we compared transcriptomes of parental cancer cells and PGCC in the absence or presence of the ASAH1 inhibitor LCL521. Results show that PGCC express less INSIG1, which downregulates cholesterol metabolism and that inhibition of ASAH1 increased HMGCR which is the rate limiting enzyme in cholesterol synthesis. Confocal microscopy revealed that ceramide and cholesterol do not colocalize. Treatment with LCL521 or simvastatin to inhibit ASAH1 or HMGCR, respectively, resulted in accumulation of ceramide at the cell surface of PGCC and prevented PGCC progeny formation. Our results suggest that similarly to inhibition of ASAH1, disruption of cholesterol signaling is a potential strategy to interfere with PGCC progeny formation.

Case report: Awake craniotomy during pregnancy for resection of glioblastoma.

Few cases have been reported of the diagnosis and treatment of glioblastoma (GB) during pregnancy. Subsequently, surgical, medical, and obstetrical management of complicated primary central nervous system malignancy in antepartum and postpartum patients remains under-investigated. The authors report the case of a 24-year-old female patient who developed generalized tonic-clonic seizures and focal neurologic deficits. MRI imaging (3T Skyra, Siemens, Erlangen, Germany) revealed an intracranial mass suspicious for malignant tumor and surgical resection under awake sedation was scheduled. The patient was incidentally found to be in her first trimester of pregnancy. Using neuronavigation, neurophysiologic monitoring, and conscious sedation the tumor was debulked successfully and histopathologic analysis confirmed giant cell glioblastoma, WHO Grade IV, 1p/19q intact, IDH wild-type, with NF1 p.Y2285fs and RB1 p.S318fs somatic mutations. Post-surgical oncologic management continued with fractioned radiotherapy and use of the Optune® device. The patient underwent uncomplicated cesarean section at 34-weeks gestation, the child remains healthy and the patient remains disease-disease free at 1-year. Thus, this case presents an approach to management of complicated GBM during first trimester pregnancy.

Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs).

BACKGROUND: Genomic instability and chemoresistance can arise in cancer due to a unique form of plasticity: that of polyploid giant cancer cells (PGCCs). These cells form under the stress of chemotherapy and have higher than diploid chromosome content. PGCCs are able to then repopulate tumors through an asymmetric daughter cell budding process. PGCCs have been observed in ovarian cancer histology, including the deadly and common form high-grade serous ovarian carcinoma (HGSC). We previously discovered that drugs which disrupt the cellular recycling process of autophagy are uniquely efficacious in pre-clinical HGSC models. While autophagy induction has been associated with PGCCs, it has never been previously investigated if autophagy modulation interacts with the PGCC life cycle and this form of tumor cell plasticity. METHODS: CAOV3 and OVCAR3 ovarian cancer cell lines were treated with carboplatin or docetaxel to induce PGCC formation. Microscopy was used to characterize and quantify PGCCs formed by chemotherapy. Two clinically available drugs that inhibit autophagy, hydroxychloroquine and nelfinavir, and a clinically available activator of autophagy, rapamycin, were employed to test the effect of these autophagy modulators on PGCC induction and subsequent colony formation from PGCCs. Crystal violet-stained colony formation assays were used to quantify the tumor-repopulating stage of the PGCC life cycle. RESULTS: Autophagy inhibitors did not prevent PGCC formation in OVCAR3 or CAOV3 cells. Rapamycin did not induce PGCC formation on its own nor did it exacerbate PGCC formation by chemotherapy. However, hydroxychloroquine prevented efficient colony formation in CAOV3 PGCCs induced by carboplatin (27% inhibition) or docetaxel (41% inhibition), as well as in OVCAR3 cells (95% and 77%, respectively). Nelfinavir similarly prevented colony formation in CAOV3 PGCCs induced by carboplatin (64% inhibition) or docetaxel (94% inhibition) as well as in OVCAR3 cells (89% and 80%, respectively). Rapamycin surprisingly also prevented PGCC colony outgrowth (52-84% inhibition). CONCLUSIONS: While the autophagy previously observed to correlate with PGCC formation is unlikely necessary for PGCCs to form, autophagy modulating drugs severely impair the ability of HGSC PGCCs to form colonies. Clinical trials which utilize hydroxychloroquine, nelfinavir, and/or rapamycin after chemotherapy may be of future interest.

Sphingolipids in embryonic development, cell cycle regulation, and stemness - Implications for polyploidy in tumors.

The aberrant biology of polyploid giant cancer cells (PGCC) includes dysregulation of the cell cycle, induction of stress responses, and dedifferentiation, all of which are likely accompanied by adaptations in biophysical properties and metabolic activity. Sphingolipids are the second largest class of membrane lipids and play important roles in many aspects of cell biology that are potentially relevant to polyploidy. We have recently shown that the function of the sphingolipid enzyme acid ceramidase (ASAH1) is critical for the ability of PGCC to generate progeny by depolyploidization but mechanisms by which sphingolipids contribute to polyploidy and generation of offspring with stem-like properties remain elusive. This review discusses the role of sphingolipids during embryonic development, cell cycle regulation, and stem cells in an effort to highlight parallels to polyploidy.

Ceramide Synthase 6 Maximizes p53 Function to Prevent Progeny Formation from Polyploid Giant Cancer Cells.

Polyploid giant cancer cells (PGCC) constitute a transiently senescent subpopulation of cancer cells that arises in response to stress. PGCC are capable of generating progeny via a primitive, cleavage-like cell division that is dependent on the sphingolipid enzyme acid ceramidase (ASAH1). The goal of this study was to understand differences in sphingolipid metabolism between non-polyploid and polyploid cancer cells to gain an understanding of the ASAH1-dependence in the PGCC population. Steady-state and flux analysis of sphingolipids did not support our initial hypothesis that the ASAH1 product sphingosine is rapidly converted into the pro-survival lipid sphingosine-1-phosphate. Instead, our results suggest that ASAH1 activity is important for preventing the accumulation of long chain ceramides such as C16-ceramide. We therefore determined how modulation of C16-ceramide, either through CerS6 or p53, a known PGCC suppressor and enhancer of CerS6-derived C16-ceramide, affected PGCC progeny formation. Co-expression of the CerS6 and p53 abrogated the ability of PGCC to form offspring, suggesting that the two genes form a positive feedback loop. CerS6 enhanced the effect of p53 by significantly increasing protein half-life. Our results support the idea that sphingolipid metabolism is of functional importance in PGCC and that targeting this signaling pathway has potential for clinical intervention.

The Uninsured are a Distinct Population in a Breast Cancer Cohort, with Unique Screening, Staging, and Outcomes.

Insurance status is a known determinant of cancer stage at diagnosis and outcome. However, insurance status can change over the course of the disease and its treatment, complicating causal analysis. Cancer registries strive to capture the insurance status of patients at diagnosis, but this is not always possible. Breast cancer poses a particular challenge for this effort, as uninsured patients become eligible for Medicaid upon the diagnosis. Thus, their insurance status may have changed from uninsured to Medicaid by the time registrars interact with treatment records. We addressed this potential blurring between categories by working with a sample of patients identified through the cancer registry of the Medical University of South Carolina to focus on determining insurance status at diagnosis whenever possible. We found that the uninsured population (32 women) was larger than the Medicaid-covered population (22 women) in a sample of patients in South Carolina, a state that did not accept the Medicaid expansion. Compared with women who carried any type of insurance, uninsured women were much more likely to find their own breast mass through palpation rather than through screening, they were diagnosed with a later stage of breast cancer at diagnosis, and their outcomes were worse. Insured women experienced significantly increased survival odds (odds ratio, 3.28) and multiple regression analysis demonstrated that the higher stages seen in uninsured women largely accounted for the poorer outcomes. These findings suggest that more research is needed to define the characteristics and disease courses unique to the breast cancer population lacking insurance prior to diagnosis.

Giants and monsters: Unexpected characters in the story of cancer recurrence.

Polyploid giant cancer cells (PGCC) constitute a dangerous subpopulation of cancer cells and are a driving force in cancer recurrence. These unique cells arise from diploid tumor cells in response to stress encountered in the tumor microenvironment or during cancer therapy. PGCC are greatly dedifferentiated, acquire pluripotency, and are able to replicate through a form of asymmetric division called neosis, which results in new populations that are themselves able to differentiate into new cell types or to re-establish tumors. Progeny tend to be more genetically unstable than the founding population due to the dysregulation required to transition through a PGCC state. Therefore, cancers that escape stressors through this mechanism tend to re-emerge with a more aggressive phenotype that is therapy resistant. This review focuses on the clinical significance of PGCC, the need for standardized nomenclature and molecular markers, as well as possible avenues to develop therapies aimed at PGCC and the process of neosis. The biology underlying the development of PGCC including cell cycle checkpoint dysregulation, stress responses, dedifferentiation, stemness and epithelial-mesenchymal transition is discussed.

Inhibition of Histone Deacetylase (HDAC) Enhances Checkpoint Blockade Efficacy by Rendering Bladder Cancer Cells Visible for T Cell-Mediated Destruction.

Inhibitory checkpoint blockade therapy is an immunomodulatory strategy that results in the restoration of T cell functions, and its efficacy depends on the recognition of tumor cells for destruction. Considering the factors at play, one could propose that anti-tumor responses will not occur if tumor cells are immunologically invisible to T cells. In this study, we tested a strategy based on the modulation of cancer cell's immunovisibility through HDAC inhibition. In a model (heterotopic and orthotopic) of mouse urothelial bladder cancer, we demonstrated that the use of intratumoral or intravesical HDACi in combination with systemic anti-PD-1 was effective at inducing curative responses with durable anti-tumor immunity capable of preventing tumor growth at a distal site. Mechanistically, we determined that protective responses were dependent on CD8 cells, but not NK cells. Of significance, in an in vitro human model, we found that fully activated T cells fail at killing bladder cancer cells unless tumor cells were pretreated with HDACi. Complementary to this observation, we found that HDACi cause gene deregulation, that results in the upregulation of genes responsible for mediating immunorecognition, NKG2D ligands and HSP70. Taken together, these data indicate that HDAC inhibition results in the elimination of the tumor cell's "invisibility cloak" that prevents T cells from recognizing and killing them. Finally, as checkpoint blockade therapy moves into the adjuvant setting, its combined use with locally administrated HDACi represents a new approach to be included in our current therapeutic treatment toolbox.

Tamoxifen is a candidate first-in-class inhibitor of acid ceramidase that reduces amitotic division in polyploid giant cancer cells-Unrecognized players in tumorigenesis.

Polyploid giant cancer cells (PGCC) represent a poorly understood, small subpopulation of tumor cells that are increasingly being recognized for their critical role in therapy resistance, metastasis, and cancer recurrence. PGCC have the potential to generate progeny through primitive or cleavage-like division, which allows them to evade antimitotic insults. We recently demonstrated that the sphingolipid enzyme acid ceramidase (ASAH1) is required for this process. Since specific ASAH1 inhibitors are not clinically available, we investigated whether tamoxifen, which interferes with ASAH1 function via off-target effects, has a potential clinical benefit independent of estrogen signaling. Our results show that tamoxifen inhibits generation of PGCC offspring in prostate cancer, glioblastoma, and melanoma cells. Analysis of two state-level cancer registries revealed that tamoxifen improves survival outcomes for second, nonbreast cancers that develop in women with early stage breast cancer. Our results suggest that tamoxifen may have a clinical benefit in a variety of cancers that is independent of estrogen signaling and could be due to its inhibition of acid ceramidase. Thus the distinct application of tamoxifen as potentially a first-in-class therapeutic that inhibits the generation of PGCC offspring should be considered in future clinical trials.

Delivery of TRAIL-expressing plasmid DNA to cancer cells in vitro and in vivo using aminoglycoside-derived polymers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand that can preferentially induce apoptosis in cancer cells over normal cells. The transmembrane form of TRAIL has been shown to elicit much stronger activity than its soluble counterpart but delivery is a potential challenge. Here, we investigated the potential of aminoglycoside-derived polymers to enhance delivery of a plasmid (pEF-TRAIL) that expresses the transmembrane form of TRAIL in order to determine the effect on cell death in vitro and tumor growth in vivo. Transgene delivery efficacy and toxicity of aminoglycoside-derived polymers was first evaluated using a GFP-expressing plasmid (pEF-GFP) at different plasmid amounts and plasmid : polymer ratios in UMUC3 bladder cancer and HeLa cervical cancer cells. Delivery of the TRAIL plasmid using aminoglycoside-derived polymers resulted in up to 60% cell death in UMUC3 and HeLa cells; TRAIL protein expression was confirmed using Western blots. TRAIL plasmid delivery resulted in a decrease in cellular procaspase-8 and an increase in TRAIL receptor DR5 levels, suggesting a role for the death receptor and caspase cascade in TRAIL-mediated apoptosis. The TRAIL plasmid did not cause cell death in normal human or mouse fibroblasts. The in vivo delivery of the TRAIL plasmid using a paromomycin-derived polymer resulted in significant reduction in tumor burden and increased survival in tumor-bearing live mice.

A Rapid Array-Based Approach to N-Glycan Profiling of Cultured Cells.

Typically, N-glycosylation studies done on cultured cells require up to millions of cells followed by lengthy preparation to release, isolate, and profile N-glycans. To overcome these limitations, we report a rapid array-based workflow for profiling N-glycan signatures from cells, adapted from imaging mass spectrometry used for on-tissue N-glycan profiling. Using this approach, N-glycan profiles from a low-density array of eight cell chambers could be reported within 4 h of completing cell culture. Approaches are demonstrated that account for background N-glycans due to serum media. Normalization procedures are shown. The method is robust and reproducible, requiring as few as 3000 cells per replicate with a 3-20% coefficient of variation to capture label-free profiles of N-glycans. Quantification by stable isotopic labeling of N-glycans in cell culture is demonstrated and adds no additional time to preparation. Utility of the method is demonstrated by measurement of N-glycan turnover rates due to induction of oxidative stress in human primary aortic endothelial cells. The developed method and ancillary tools serve as a foundational launching point for rapid profiling of N-glycans ranging from high-density arrays down to single cells in culture.

Genetic and pharmacological inhibition of acid ceramidase prevents asymmetric cell division by neosis.

Radiation treatment failure or relapse after initial response to chemotherapy presents significant clinical challenges in cancer patients. Escape from initial courses of treatment can involve reactivation of embryonic developmental stages, with the formation of polynuclear giant cancer cells (PGCCs). This strategy of dedifferentiation can insulate cancer cells from a variety of treatments and allows a residual subpopulation to reestablish tumors after treatment. Using radiation or docetaxel chemotherapy, we generated PGCCs from prostate cancer cells. Here, we show that expression of acid ceramidase (ASAH1), an enzyme in the sphingolipid pathway linked to therapy resistance and poor outcomes, is elevated in PGCCs. Targeting ASAH1 with shRNA or treatment with the ASAH1 inhibitor, LCL-521, did not impair the formation of PGCCs, but prevented the formation of PGCC progeny that arise through an asymmetric cell division called neosis. Similar results were obtained in lung cancer cells that had been exposed to radiation or cisplatin chemotherapy as stressors. In summary, our data suggest that endoreplication occurs independent of ASAH1 while neosis is ASAH1-dependent in both prostate and lung cancer cells. Because ASAH1 knockout is embryonic lethal but not deleterious to adult animals, targeting this enzyme has the potential to be highly specific to cells undergoing the dedifferentiation process to escape cancer treatments. Pharmacological inhibition of ASAH1 is a potentially powerful strategy to eliminate cells that could otherwise serve as seed populations for recurrence.

Expression of the SNAI2 transcriptional repressor is regulated by C16-ceramide.

Ceramide synthase 6 (CerS6) is an enzyme that preferentially generates pro-apoptotic C16-ceramide in the sphingolipid metabolic pathway. Reduced expression of CerS6 has been associated with apoptosis resistance and recent studies point to a role for CerS6 in epithelial mesenchymal transition (EMT). Because cells that undergo EMT are also more resistant to apoptosis, we hypothesized that reduced expression of CerS6 could induce changes that are associated with EMT. We found that shRNA-mediated knockdown of CerS6 increases expression of the EMT transcription factor SNAI2 but not SNAI1 or TWIST. Treatment with C6-ceramide nanoliposomes (CNL) resulted in a preferential increase in C16-ceramide and suppressed SNAI2 transcriptional activation and protein expression. The increase in C16-ceramide following CNL treatment was dependent on CerS activity and occurred even when CerS6 shRNA was expressed. shRNA against CerS5, which like CerS6 preferentially generates C16-ceramide, also decreased transcriptional activation of SNAI2, suggesting a role for C16-ceramide rather than a specific enzyme in the regulation of this transcription factor. While loss of CerS6 has been associated with apoptosis resistance, we found that cells lacking this protein are more susceptible to the effects CNL. In summary, our study identifies SNAI2 as a novel target whose expression can be influenced by C16-ceramide levels. The potential of CNL to suppress SNAI2 expression has important clinical implications, since elevated expression of this transcription factor has been associated with an aggressive phenotype or poor outcomes in several types of solid tumors.

Endoplasmic reticulum stress, autophagic and apoptotic cell death, and immune activation by a natural triterpenoid in human prostate cancer cells.

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC50 s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 + T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.

Receptor-interacting Ser/Thr kinase 1 (RIPK1) and myosin IIA-dependent ceramidosomes form membrane pores that mediate blebbing and necroptosis.

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.