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.

SWAN pathway-network identification of common aneuploidy-based oncogenic drivers.

Haploinsufficiency drives Darwinian evolution. Siblings, while alike in many aspects, differ due to monoallelic differences inherited from each parent. In cancer, solid tumors exhibit aneuploid genetics resulting in hundreds to thousands of monoallelic gene-level copy-number alterations (CNAs) in each tumor. Aneuploidy patterns are heterogeneous, posing a challenge to identify drivers in this high-noise genetic environment. Here, we developed Shifted Weighted Annotation Network (SWAN) analysis to assess biology impacted by cumulative monoallelic changes. SWAN enables an integrated pathway-network analysis of CNAs, RNA expression, and mutations via a simple web platform. SWAN is optimized to best prioritize known and novel tumor suppressors and oncogenes, thereby identifying drivers and potential druggable vulnerabilities within cancer CNAs. Protein homeostasis, phospholipid dephosphorylation, and ion transport pathways are commonly suppressed. An atlas of CNA pathways altered in each cancer type is released. These CNA network shifts highlight new, attractive targets to exploit in solid tumors.

Autophagy gene haploinsufficiency drives chromosome instability, increases migration, and promotes early ovarian tumors.

Autophagy, particularly with BECN1, has paradoxically been highlighted as tumor promoting in Ras-driven cancers, but potentially tumor suppressing in breast and ovarian cancers. However, studying the specific role of BECN1 at the genetic level is complicated due to its genomic proximity to BRCA1 on both human (chromosome 17) and murine (chromosome 11) genomes. In human breast and ovarian cancers, the monoallelic deletion of these genes is often co-occurring. To investigate the potential tumor suppressor roles of two of the most commonly deleted autophagy genes in ovarian cancer, BECN1 and MAP1LC3B were knocked-down in atypical (BECN1+/+ and MAP1LC3B+/+) ovarian cancer cells. Ultra-performance liquid chromatography mass-spectrometry metabolomics revealed reduced levels of acetyl-CoA which corresponded with elevated levels of glycerophospholipids and sphingolipids. Migration rates of ovarian cancer cells were increased upon autophagy gene knockdown. Genomic instability was increased, resulting in copy-number alteration patterns which mimicked high grade serous ovarian cancer. We further investigated the causal role of Becn1 haploinsufficiency for oncogenesis in a MISIIR SV40 large T antigen driven spontaneous ovarian cancer mouse model. Tumors were evident earlier among the Becn1+/- mice, and this correlated with an increase in copy-number alterations per chromosome in the Becn1+/- tumors. The results support monoallelic loss of BECN1 as permissive for tumor initiation and potentiating for genomic instability in ovarian cancer.

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.

Aneuploidy: An Opportunity Within Single-Cell RNA Sequencing Analysis.

Single-cell sequencing data has transformed the understanding of biological heterogeneity. While many flavors of single-cell sequencing have been developed, single-cell RNA sequencing (scRNA-seq) is currently the most prolific form in published literature. Bioinformatic analysis of differential biology within the population of cells studied relies on inferences and grouping of cells due to the spotty nature of data within individual cell scRNA-seq gene counts. One biologically relevant variable is readily inferred from scRNA-seq gene count tables regardless of individual gene representation within single cells: aneuploidy. Since hundreds of genes are present on chromosome arms, high-quality inferences of aneuploidy can be made from scRNA-seq datasets. This viewpoint summarizes how utilization of these bioinformatic pipelines can benefit scRNA-seq studies, particularly in oncology wherein aneuploidy is both rampant and a hallmark of the studied disease. Awareness and use of these analytical pipelines will improve each field's ability to understand the studied diseases. Authors are encouraged to attempt these aneuploid analyses when reporting scRNA-seq data, much like copy-number variants are commonly reported in bulk genome sequencing data.

Lifespan extension conferred by endoplasmic reticulum secretory pathway deficiency requires induction of the unfolded protein response.

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.

Elevated proteasome capacity extends replicative lifespan in Saccharomyces cerevisiae.

Aging is characterized by the accumulation of damaged cellular macromolecules caused by declining repair and elimination pathways. An integral component employed by cells to counter toxic protein aggregates is the conserved ubiquitin/proteasome system (UPS). Previous studies have described an age-dependent decline of proteasomal function and increased longevity correlates with sustained proteasome capacity in centenarians and in naked mole rats, a long-lived rodent. Proof for a direct impact of enhanced proteasome function on longevity, however, is still lacking. To determine the importance of proteasome function in yeast aging, we established a method to modulate UPS capacity by manipulating levels of the UPS-related transcription factor Rpn4. While cells lacking RPN4 exhibit a decreased non-adaptable proteasome pool, loss of UBR2, an ubiquitin ligase that regulates Rpn4 turnover, results in elevated Rpn4 levels, which upregulates UPS components. Increased UPS capacity significantly enhances replicative lifespan (RLS) and resistance to proteotoxic stress, while reduced UPS capacity has opposing consequences. Despite tight transcriptional co-regulation of the UPS and oxidative detoxification systems, the impact of proteasome capacity on lifespan is independent of the latter, since elimination of Yap1, a key regulator of the oxidative stress response, does not affect lifespan extension of cells with higher proteasome capacity. Moreover, since elevated proteasome capacity results in improved clearance of toxic huntingtin fragments in a yeast model for neurodegenerative diseases, we speculate that the observed lifespan extension originates from prolonged elimination of damaged proteins in old mother cells. Epistasis analyses indicate that proteasome-mediated modulation of lifespan is at least partially distinct from dietary restriction, Tor1, and Sir2. These findings demonstrate that UPS capacity determines yeast RLS by a mechanism that is distinct from known longevity pathways and raise the possibility that interventions to promote enhanced proteasome function will have beneficial effects on longevity and age-related disease in humans.

MYC and HSF1 Cooperate to Drive PLK1 inhibitor Sensitivity in High Grade Serous Ovarian Cancer.

Ovarian cancer is a deadly female cancer with high rates of recurrence. The primary treatment strategy for patients is platinum-based therapy regimens that almost universally develop resistance. Consequently, new therapeutic avenues are needed to overcome the plateau that current therapies have on patient outcomes. We describe a gene amplification involving both HSF1 and MYC, wherein these two genes on chromosome 8q are co-amplified in over 7% of human tumors that is enriched to over 30% of patients with ovarian cancer. We further found that HSF1 and MYC transcriptional activity is correlated in human tumors and ovarian cancer cell lines, suggesting they may cooperate in ovarian cancer cells. CUT&RUN for HSF1 and MYC in co-amplified ovarian cancer cells revealed that HSF1 and MYC have overlapping binding at a substantial number of locations throughout the genome where their binding peaks are near identical. Consistent with these data, a protein-protein interaction between HSF1 and MYC was detected in ovarian cancer cells, implying these two transcription factors have a molecular cooperation. Further supporting their cooperation, growth of HSF1-MYC co-amplified ovarian cancer cells were found to be dependent on both HSF1 and MYC. In an attempt to identify a therapeutic target that could take advantage of this dependency on both HSF1 and MYC, PLK1 was identified as being correlated with HSF1 and MYC in primary human tumor specimens, consistent with a previously established effect of PLK1 on HSF1 and MYC protein levels. Targeting PLK1 with the compound volasertib (BI-6727) revealed a greater than 200-fold increased potency of volasertib in HSF1-MYC co-amplified ovarian cancer cells compared to ovarian cancer cells wild-type HSF1 and MYC copy number, which extended to several growth assays, including spheroid growth. Volasertib, and other PLK1 inhibitors, have not shown great success in clinical trials and this study suggests that targeting PLK1 may be viable in a precision medicine approach using HSF1-MYC co-amplification as a biomarker for response.

MYC is sufficient to generate mid-life high-grade serous ovarian and uterine serous carcinomas in a p53-R270H mouse model.

Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment is understood. However, previous GEMMs of high-grade serous ovarian cancer (HGSOC) have had to utilize genetics rarely or never found in human HGSOC to yield ovarian cancer within the lifespan of a mouse. MYC, an oncogene, is amongst the most amplified genes in HGSOC, but it has not previously been utilized to drive HGSOC GEMMs. We coupled Myc and dominant negative mutant p53-R270H with a fallopian tube epithelium-specific promoter Ovgp1 to generate a new GEMM of HGSOC. Female mice developed lethal cancer at an average of 15.1 months. Histopathological examination of mice revealed HGSOC characteristics including nuclear p53 and nuclear MYC in clusters of cells within the fallopian tube epithelium and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the fallopian tube epithelium (FTE). Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Taken together, these results demonstrate the Myc and Trp53-R270H transgene was able to recapitulate many phenotypic hallmarks of HGSOC through the utilization of strictly human-mimetic genetic hallmarks of HGSOC. This new mouse model enables further exploration of ovarian cancer pathogenesis, particularly in the 50% of HGSOC which lack homology directed repair mutations. Histological and transcriptomic findings are consistent with the hypothesis that uterine serous cancer may originate from the fallopian tube epithelium.

Variants in the Kallikrein Gene Family and Hypermobile Ehlers-Danlos Syndrome.

Hypermobile Ehlers-Danlos syndrome (hEDS) is a common heritable connective tissue disorder that lacks a known genetic etiology. To identify genetic contributions to hEDS, whole exome sequencing was performed on families and a cohort of sporadic hEDS patients. A missense variant in Kallikrein-15 (KLK15 p. Gly226Asp ) , segregated with disease in two families and genetic burden analyses of 197 sporadic hEDS patients revealed enrichment of variants within the Kallikrein gene family. To validate pathogenicity, the variant identified in familial studies was used to generate knock-in mice. Consistent with our clinical cohort, Klk15 G224D/+ mice displayed structural and functional connective tissue defects within multiple organ systems. These findings support Kallikrein gene variants in the pathogenesis of hEDS and represent an important step towards earlier diagnosis and better clinical outcomes.

End-of-life cell cycle arrest contributes to stochasticity of yeast replicative aging.

There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in life span that can be modeled by the Gompertz-Makeham law of mortality. Here, we report that within genetically identical haploid and diploid wild-type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean life span (25.6 vs. 35.6) and larger coefficient of variance with respect to individual life span (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten life span and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.

Protocol for mapping double-stranded DNA break sites across the genome with translocation capture sequencing.

Translocation sequencing can be used to assess mechanisms of DNA repair and identify genome-wide double-strand breaks (DSBs) accessible to DNA repair machinery. Here, we present a protocol for mapping double-strand DNA break sites across the genome with translocation capture sequencing. Bait DSBs are introduced using a Cas9 nuclease and repaired by the host cell, connecting bait DSBs to other DSBs. Repair sites are detected by isolating bait site DNA, cleaving normal sequence to enrich off-site repair, and next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Switonski et al. (2021).1.

Autophagy dictates sensitivity to PRMT5 inhibitor in breast cancer.

Protein arginine methyltransferase 5 (PRMT5) catalyzes mono-methylation and symmetric di-methylation on arginine residues and has emerged as a potential antitumor target with inhibitors being tested in clinical trials. However, it remains unknown how the efficacy of PRMT5 inhibitors is regulated. Here we report that autophagy blockage enhances cellular sensitivity to PRMT5 inhibitor in triple negative breast cancer cells. Genetic ablation or pharmacological inhibition of PRMT5 triggers cytoprotective autophagy. Mechanistically, PRMT5 catalyzes monomethylation of ULK1 at R532 to suppress ULK1 activation, leading to attenuation of autophagy. As a result, ULK1 inhibition blocks PRMT5 deficiency-induced autophagy and sensitizes cells to PRMT5 inhibitor. Our study not only identifies autophagy as an inducible factor that dictates cellular sensitivity to PRMT5 inhibitor, but also unearths a critical molecular mechanism by which PRMT5 regulates autophagy through methylating ULK1, providing a rationale for the combination of PRMT5 and autophagy inhibitors in cancer therapy.

Combination of Autophagy Selective Therapeutics With Doxil: An Assessment of Pathological Toxicity.

Background: Combination therapy of targeted drugs in cancer treatment is a field in constant flux, with research balancing side effects with efficacy. Efficacy from combination therapy is improved either through synthetic lethality or through prevention of recurrent clones. Previous research has shown (hydroxy-)chloroquine is insufficient to disrupt autophagy in tumors. Hence, either combinations or novel autophagy agents are desired. In vivo studies of ovarian cancer have revealed that chloroquine can be combined with up to four other autophagy drugs to suppress ovarian cancer growth. While cancer efficacy is now established for the autophagy drug combination, it is unclear what toxicities may require monitoring in human trials. Additive toxicity with chemotherapy is also unknown. Methods: To address toxicity in more depth than previous weight-monitoring studies, biochemical and histopathology studies were performed. Mouse groups were treated with autophagy drugs for 2 weeks, with or without the chemotherapy Doxil. After the last dose, mice were processed for blood biochemistry, white blood cell markers, and histopathology. Results: Data from a comprehensive blood biochemistry panel, flow cytometric measurements of blood cell markers, and histopathology are herein reported. While Doxil presented clear bone marrow and immunologic toxicity, autophagy drugs were overall less toxic and more variable in their presentation of potential toxicities. Only minor additive effects of autophagy drugs with Doxil were observed. Conclusion: Combinations of autophagy drugs may be considered for therapy in human oncology trials, with possible side effects to monitor informed by these murine pre-clinical data.

Arginine methylation of BRD4 by PRMT2/4 governs transcription and DNA repair.

BRD4 functions as an epigenetic reader and plays a crucial role in regulating transcription and genome stability. Dysregulation of BRD4 is frequently observed in various human cancers. However, the molecular details of BRD4 regulation remain largely unknown. Here, we report that PRMT2- and PRMT4-mediated arginine methylation is pivotal for BRD4 functions on transcription, DNA repair, and tumor growth. Specifically, PRMT2/4 interacts with and methylates BRD4 at R179, R181, and R183. This arginine methylation selectively controls a transcriptional program by promoting BRD4 recruitment to acetylated histones/chromatin. Moreover, BRD4 arginine methylation is induced by DNA damage and thereby promotes its binding to chromatin for DNA repair. Deficiency in BRD4 arginine methylation significantly suppresses tumor growth and sensitizes cells to BET inhibitors and DNA damaging agents. Therefore, our findings reveal an arginine methylation-dependent regulatory mechanism of BRD4 and highlight targeting PRMT2/4 for better antitumor effect of BET inhibitors and DNA damaging agents.

Profiling of RNA-binding Proteins Interacting With Glucagon and Adipokinetic Hormone mRNAs.

OBJECTIVE: Glucagon in mammals and its homolog (adipokinetic hormone [AKH] in Drosophila melanogaster) are peptide hormones which regulate lipid metabolism by breaking down triglycerides. Although regulatory mechanisms of glucagon and AKH expression have been widely studied, post-transcriptional gene expression of glucagon has not been investigated thoroughly. In this study, we aimed to profile proteins binding with Gcg messenger RNA (mRNA) in mouse and Akh mRNA in Drosophila. METHODS: Drosophila Schneider 2 (S2) and mouse 3T3-L1 cell lysates were utilized for affinity pull down of Akh and Gcg mRNA respectively using biotinylated anti-sense DNA oligoes against target mRNAs. Mass spectrometry and computational network analysis revealed mRNA-interacting proteins residing in functional proximity. RESULTS: We observed that 1) 91 proteins interact with Akh mRNA from S2 cell lysates, 2) 34 proteins interact with Gcg mRNA from 3T3-L1 cell lysates. 3) Akh mRNA interactome revealed clusters of ribosomes and known RNA-binding proteins (RBPs). 4) Gcg mRNA interactome revealed mRNA-binding proteins including Plekha7, zinc finger protein, carboxylase, lipase, histone proteins and a cytochrome, Cyp2c44. 5) Levels of Gcg mRNA and its interacting proteins are elevated in skeletal muscles isolated from old mice compared to ones from young mice. CONCLUSION: Akh mRNA in S2 cells are under active translation in a complex of RBPs and ribosomes. Gcg mRNA in mouse precursor adipocyte is in a condition distinct from Akh mRNA due to biochemical interactions with a subset of RBPs and histones. We anticipate that our study contributes to investigating regulatory mechanisms of Gcg and Akh mRNA decay, translation, and localization.

Altered H3 histone acetylation impairs high-fidelity DNA repair to promote cerebellar degeneration in spinocerebellar ataxia type 7.

A common mechanism in inherited ataxia is a vulnerability of DNA damage. Spinocerebellar ataxia type 7 (SCA7) is a CAG-polyglutamine-repeat disorder characterized by cerebellar and retinal degeneration. Polyglutamine-expanded ataxin-7 protein incorporates into STAGA co-activator complex and interferes with transcription by altering histone acetylation. We performed chromatic immunoprecipitation sequencing ChIP-seq on cerebellum from SCA7 mice and observed increased H3K9-promoter acetylation in DNA repair genes, resulting in increased expression. After detecting increased DNA damage in SCA7 cells, mouse primary cerebellar neurons, and patient stem-cell-derived neurons, we documented reduced homology-directed repair (HDR) and single-strand annealing (SSA). To evaluate repair at endogenous DNA in native chromosome context, we modified linear amplification-mediated high-throughput genome-wide translocation sequencing and found that DNA translocations are less frequent in SCA7 models, consistent with decreased HDR and SSA. Altered DNA repair function in SCA7 may predispose the subject to excessive DNA damage, leading to neuron demise and highlights DNA repair as a therapy target.

Single-cell analysis of copy-number alterations in serous ovarian cancer reveals substantial heterogeneity in both low- and high-grade tumors.

Unusually high aneuploidy is a hallmark of epithelial serous ovarian cancer (SOC). Previous analyses have focused on aneuploidy on average across all tumor cells. With the expansion of single-cell sequencing technologies, however, an analysis of copy number heterogeneity cell-to-cell is now technically feasible. Here, we describe an analysis of single-cell RNA sequencing (scRNA-seq) data to infer arm-level aneuploidy in individual serous ovarian cancer cells. By first clustering high-quality sequenced epithelial versus non-epithelial cells, high-confidence tumor cell populations were identified. InferCNV was used to predict segmented copy-number alterations (CNAs), which were then used to determine arm-level aneuploidy at the single-cell level. Control comparisons of normal cells to normal cells showed zero arm-level aneuploidy, whereas a median of four aneuploid events were detectable in cancer cells. A heterogeneity analysis of high-grade tumor cells compared to low-grade tumor cells showed similar levels of cell-to-cell variation between cancer grades. Metastatic tumors potentially showed selection pressure with reduced cell-to-cell variation compared to cells from primary tumors. Minor cell populations with CNAs similar to metastatic cells were identified within the matched primary tumors. Taken together, these results provide a minimum estimate for single-cell aneuploidy in serous ovarian cancer and demonstrate the utility of single-cell sequencing for CNA analysis.

Treating the Disease, Not the Symptom: Beyond NSAIDs.

Mitigating inflammation is clearly important in cancer prevention and control. Traditionally, pharmaceuticals have taken the lead in this problem. In an attempt to 'head them off at the pass', this Forum takes a hard look at the concept of 'better living through chemicals' and limiting proinflammatory chemicals entering the body.

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.