Pluripotent stem cells for target organ developmental toxicity testing.

Prenatal developmental toxicity research focuses on understanding potential adverse effects of environmental agents, drugs, and chemicals on the development of embryos and fetuses. Traditional methods involve animal testing, but ethical concerns and the need for human-relevant models have prompted the exploration of alternatives. Pluripotent stem cells (PSCs) are versatile cells with the unique ability to differentiate into any cell type, serving as a foundational tool for studying human development. Two-dimensional (2D) PSC models are often chosen for their ease of use and reproducibility for high-throughput screening. However, they lack the complexity of an in vivo environment. Alternatively, three-dimensional (3D) PSC models, such as organoids, offer tissue architecture and intercellular communication more reminiscent of in vivo conditions. However, they are complicated to produce and analyze, usually requiring advanced and expensive techniques. This review discusses recent advances in the use of human PSCs differentiated into brain and heart lineages and emerging tools and methods that can be combined with PSCs to help address important scientific questions in the area developmental toxicology. These advancements and new approach methods align with the push for more relevant and predictive developmental toxicity assessment, combining innovative techniques with organoid models to advance regulatory decision-making.

Ferroptosis-Mediated Cell Death Induced by NCX4040, The Non-Steroidal Nitric Oxide Donor, in Human Colorectal Cancer Cells: Implications in Therapy.

Our recent studies show that the treatment of human ovarian tumor cells with NCX4040 results in significant depletions of cellular glutathione, the formation of reactive oxygen/nitrogen species and cell death. NCX4040 is also cytotoxic to several human colorectal cancer (CRC) cells in vitro and in vivo. Here, we examined the ferroptosis-dependent mechanism(s) of cytotoxicity of NCX4040 in HT-29 and K-RAS mutant HCT 116 colon cell lines. Ferroptosis is characterized by the accumulation of reactive oxygen species (ROS) within the cell, leading to an iron-dependent oxidative stress-mediated cell death. However, its relevance in the mechanism of NCX4040 cytotoxicity in CRCs is not known. We found that NCX4040 generates ROS in CRC cells without any depletion of cellular GSH. Combinations of NCX4040 with erastin (ER) or RSL3 (RAS-selective lethal 3), known inducers of ferroptosis, enhanced CRC death. In contrast, ferrostatin-1, an inhibitor of ferroptosis, significantly inhibited NCX4040-induced cell death. Treatment of CRC cells with NCX4040 resulted in the induction of lipid peroxidation in a dose- and time-dependent manner. NCX4040 treatment induced several genes related to ferroptosis (e.g., CHAC1, GPX4 and NOX4) in both cell lines. Metabolomic studies also indicated significant increases in both lipid and energy metabolism following the drug treatment in HT-29 and HCT 116 cells. These observations strongly suggest that NCX4040 causes the ferroptosis-mediated cell death of CRC cells. Furthermore, combinations of NCX4040 and ER or RSL3 may contribute significantly to the treatment of CRC, including those that are difficult to treat due to the presence of Ras mutations in the clinic. NCX4040-induced ferroptosis may also be a dynamic form of cell death for the treatment of other cancers.

Cardiac Development in the Presence of Cadmium: An in Vitro Study Using Human Embryonic Stem Cells and Cardiac Organoids.

BACKGROUND: Exposure to cadmium (Cd) is associated with cardiovascular diseases. Maternal Cd exposure is a significant risk factor for congenital heart disease. However, mechanisms of Cd on developmental cardiotoxicity are not well defined. OBJECTIVES: We evaluated the effects of Cd on the different stages (mesoderm, cardiac induction, cardiac function) of cardiac development using an early embryo development in vitro model and two- or three-dimensional (2- or 3D) cardiomyocyte and cardiac organoid formation models mimicking early cardiac development. METHODS: Embryonic stem cells (ESCs) form 3D aggregates, called embryoid bodies, that recapitulate events involved with early embryogenesis (e.g., germ layer formation). This model was used for early germ layer formation and signaling pathway identification. The 2D cardiomyocyte differentiation from the NKX2-5eGFP/w human ESCs model was used to explore the effects of Cd exposure on cardiomyocyte formation and to model mesoderm differentiation and cardiac induction, allowing us to explore different developmental windows of Cd toxicity. The 3D cardiac organoid model was used in evaluating the effects of Cd exposure on contractility and cardiac development. RESULTS: Cd (0.6µM; 110 ppb) lowered the differentiation of embryoid bodies to mesoderm via suppression of Wnt/ß-catenin-signaling pathways. During early mesoderm induction, the mesoderm-associated transcription factors MESP1 and EOMES showed a transient up-regulation, which decreased later in the cardiac induction stage. Cd (0.15µM) lowered mesoderm formation and cardiac induction through suppression of the transcription factors and mesoderm marker genes HAND1, SNAI2, HOPX, and the cardiac-specific genes NKX2-5, GATA4, troponin T, and alpha-actinin. In addition, Cd-induced histone modifications for both gene activation (H3K4me3) and repression (H3K27me3), which play vital roles in regulating mesoderm commitment markers. The effects of Cd inhibition on cardiomyocyte differentiation were confirmed in 3D cardiac organoids. DISCUSSION: In conclusion, using a human ESC-derived 2D/3D in vitro differentiation model system and cardiac organoids, we demonstrated that low-dose Cd suppressed mesoderm formation through mesoderm gene histone modification, thus inhibiting cardiomyocyte differentiation and cardiac induction. The studies provide valuable insights into cellular events and molecular mechanisms associated with Cd-induced congenital heart disease. https://doi.org/10.1289/EHP11208.

Stem Cells as Target for Prostate cancer Therapy: Opportunities and Challenges.

Cancer stem cells (CSCs) and cells in a cancer stem cell-like (CSCL) state have proven to be responsible for tumor initiation, growth, and relapse in Prostate Cancer (PCa) and other cancers; therefore, new strategies are being developed to target such cellular populations. TLR3 activation-based immunotherapy using Polyinosinic:Polycytidylic acid (PIC) has been proposed to be used as a concomitant strategy to first-line treatment. This strategy is based on the induction of apoptosis and an inflammatory response in tumor cells. In combination with retinoids like 9cRA, this treatment can induce CSCs differentiation and apoptosis. A limitation in the use of this combination is the common decreased expression of TLR3 and its main positive regulator p53. observed in many patients suffering of different cancer types such as PCa. Importantly, human exposure to certain toxicants, such as iAs, not only has proven to enrich CSCs population in an in vitro model of human epithelial prostate cells, but additionally, it can also lead to a decreased p53, TLR3 and RA receptor (RARß), expression/activation and thus hinder this treatment efficacy. Therefore, here we point out the relevance of evaluating the TLR3 and P53 status in PCa patients before starting an immunotherapy based on the use of PIC +9cRA to determine whether they will be responsive to treatment. Additionally, the use of strategies to overcome the lower TLR3, RARß or p53 expression in PCa patients, like the inclusion of drugs that increase p53 expression, is encouraged, to potentiate the use of PIC+RA based immunotherapy in these patients.

Molecular Mechanisms of Cytotoxicity of NCX4040, the Non-Steroidal Anti-Inflammatory NO-Donor, in Human Ovarian Cancer Cells.

NCX4040, the non-steroidal anti-inflammatory-NO donor, is cytotoxic to several human tumors, including ovarian tumor cells. We have found that NCX4040 is also cytotoxic against both OVCAR-8 and its adriamycin resistant (NCI/ADR-RES) tumor cell lines. Here, we have examined mechanism(s) for the cytotoxicity of NCX4040 in OVCAR-8 and NCI/ADR-RES cell lines. We found that NCX4040 induced significant apoptosis in both cell lines. Furthermore, NCX4040 treatment caused significant depletion of cellular glutathione, causing oxidative stress due to the formation of reactive oxygen/nitrogen species (ROS/RNS). Significantly more ROS/RNS were detected in OVCAR-8 cells than in NCI/ADR-RES cells which may have resulted from increased activities of SOD, glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. NCX4040 treatment resulted in the formation of double-strand DNA breaks in both cells; however, more of these DNA breaks were detected in OVCAR-8 cells. RT-PCR studies indicated that NCX4040-induced DNA damage was not repaired as efficiently in NCI/ADR-RES cells as in OVCAR-8 cells which may lead to a differential cell death. Pretreatment of OVCAR-8 cells with N-acetylcysteine (NAC) significantly decreased cytotoxicity of NCX4040 in OVCAR-8 cells; however, NAC had no effects on NCX4040 cytotoxicity in NCI/ADR-RES cells. In contrast, FeTPPS, a peroxynitrite scavenger, completely blocked NCX4040-induced cell death in both cells, suggesting that NCX4040-induced cell death could be mediated by peroxynitrite formed from NCX4040 following cellular metabolism.

Gene Expression Profiling Elucidates Cellular Responses to NCX4040 in Human Ovarian Tumor Cells: Implications in the Mechanisms of Action of NCX4040.

The nitric oxide donor, NCX4040 is a non-steroidal anti-inflammatory-NO donor and has been shown to be extremely cytotoxic to a number of human tumors, including ovarian tumors cells. We have found that NCX4040 is cytotoxic against both OVCAR-8 and its adriamycin-selected OVCAR-8 variant (NCI/ADR-RES) tumor cell lines. While the mechanism of action of NCX4040 is not entirely clear, we as well as others have shown that NCX4040 generates reactive oxygen species (ROS) and induces DNA damage in tumor cells. Recently, we have reported that NCX4040 treatment resulted in a significant depletion of cellular glutathione, and formation of both reactive oxygen and nitrogen species (ROS/RNS), resulting in oxidative stress in these tumor cells. Furthermore, our results indicated that more ROS/RNS were generated in OVCAR-8 cells than in NCI/ADR-RES cells due to increased activities of superoxide dismutase (SOD), glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. Further studies suggested that NCX4040-induced cell death may be mediated by peroxynitrite formed from NCX4040 in cells. In this study we used microarray analysis following NCX4040 treatment of both OVCAR-8 and its ADR-resistant variant to identify various molecular pathways involved in NCX4040-induced cell death. Here, we report that NCX4040 treatment resulted in the differential induction of oxidative stress genes, inflammatory response genes (TNF, IL-1, IL-6 and COX2), DNA damage response and MAP kinase response genes. A mechanism of tumor cell death is proposed based on our findings where oxidative stress is induced by NCX4040 from simultaneous induction of NOX4, TNF-α and CHAC1 in tumor cell death.

Cadmium Exposure and Ovarian Reserve in Women Aged 35-49 Years: The Impact on Results From the Creatinine Adjustment Approach Used to Correct for Urinary Dilution.

Cadmium is toxic to the ovaries in animal studies, but its association with diminished ovarian reserve in women is not established. We investigated urinary cadmium, a biomarker of long-term exposure, in relation to diminished ovarian reserve, as indicated by elevated serum follicle-stimulating hormone concentrations (≥10 IU/L), in women aged 35-49 years (unweighted n = 1,681). Using data from the Third National Health and Nutrition Examination Survey (1988-1994), we conducted Poisson regression to estimate adjusted relative risks and 95% confidence intervals. Because the best approach to correcting for urinary dilution in spot samples with creatinine remains controversial, we employed 3 approaches: standardization, covariate adjustment, and covariate-adjusted standardization. Our data suggested a modest association with standardization (highest quartile vs. lowest: relative risk (RR) = 1.3, 95% confidence interval (CI): 0.8, 1.9; P for trend = 0.06) and covariate-adjusted standardization (highest quartile vs. lowest: RR = 1.3, 95% CI: 0.9, 1.9; P for trend = 0.05) and a stronger association with covariate adjustment (highest quartile vs. lowest: RR = 1.8, 95% CI: 1.2, 2.9; P for trend = 0.01). The stronger association with covariate adjustment may reflect bias from conditioning on urinary creatinine, a collider in the hypothesized causal pathway. We conclude that cadmium may contribute to ovarian aging in women and that careful consideration of the creatinine adjustment approach is needed to minimize bias.

Depot Medroxyprogesterone Acetate Use and Blood Lead Levels in a Cohort of Young Women.

BACKGROUND: Injectable contraceptive use is common, with 74 million users worldwide. Use of the injectable contraceptive depot medroxyprogesterone acetate (DMPA) is associated with bone mineral density loss. We hypothesize that increased bone resorption with DMPA use allows for mobilization of the toxic metal lead stored in bone to blood, presenting users with increased systemic exposure to lead. OBJECTIVE: The objective of our study was to investigate the association between current DMPA use and blood lead concentrations. METHODS: We conducted a cross-sectional analysis using enrollment data from the Study of Environment, Lifestyle & Fibroids (SELF), a cohort of 1,693 African-American women who were 23-35 years of age. Data on DMPA use were collected by computer-assisted telephone interview. Blood lead concentrations were measured in whole blood samples among 1,548 participants (91% of cohort). We estimated the adjusted percent difference in blood lead concentrations and 95% confidence intervals (CI) between current DMPA users and nonusers using multivariable linear regression. RESULTS: Geometric mean blood lead concentration was 0.69µg/dL (95% CI: 0.67, 0.71). After adjustment, current DMPA users (7% of cohort) had blood lead concentrations that were 18% higher than those of nonusers (95% CI: 8%, 29%). Similar associations were observed with additional analyses to assess for potential bias from smoking, DMPA-induced amenorrhea, use of estrogen-containing contraceptives, having given birth in the prior year, and history of medical conditions or current medication use associated with bone loss. DISCUSSION: Our results indicate that current DMPA use is associated with increased blood lead concentrations. Further research, particularly in populations highly exposed to lead, is warranted to consider tradeoffs between the adverse effects of lead on human health and the importance of DMPA as a contraceptive option to prevent unintended pregnancy. https://doi.org/10.1289/EHP7017.

Elucidation of Mechanisms of Topotecan-Induced Cell Death in Human Breast MCF-7 Cancer Cells by Gene Expression Analysis.

Topotecan is a clinically active anticancer agent for the management of various human tumors. While the principal mechanism of tumor cell killing by topotecan is due to its interactions with topoisomerase I and formation of DNA double-strand breaks, recent studies suggest that mechanisms involving generation of reactive free radicals and induction of oxidative stress may play a significant role in topotecan-dependent tumor cell death. We have shown that topotecan generates a topotecan radical following one-electron oxidation by a peroxidase-hydrogen peroxide system which reacts with reduced glutathione and cysteine, forming the glutathiyl and cysteinyl radicals, respectively. While little is known how these events are involved in topotecan-induced tumor cell death, we have now examined the effects of topotecan short (1 h) and long (24 h) exposure on global gene expression patterns using gene expression microarray analysis in human breast MCF-7 cancer cells, a wild-type p53 containing cell line. We show here that topotecan treatment significantly down-regulated estrogen receptor alpha (ERα/ESR1) and antiapoptotic BCL2 genes in addition to many other p53-regulated genes. Furthermore, 8-oxoguanine DNA glycosylase (OGG1), ferredoxin reductase (FDXR), methionine sulfoxide reductase (MSR), glutathione peroxidases (GPx), and glutathione reductase (GSR) genes were also differentially expressed by topotecan treatment. The differential expression of these genes was observed in a wild-type p53-containing breast ZR-75-1 tumor cell line following topotecan treatment. The involvement of reactive oxygen free radical sensor genes, the oxidative DNA damage (OGG1) repair gene and induction of pro-apoptotic genes suggest that reactive free radical species play a role in topotecan-induced tumor cell death.

KRAS-retroviral fusion transcripts and gene amplification in arsenic-transformed, human prostate CAsE-PE cancer cells.

CAsE-PE cells are an arsenic-transformed, human prostate epithelial line containing oncogenic mutations in KRAS compared to immortalized, normal KRAS parent cells, RWPE-1. We previously reported increased copy number of mutated KRAS in CAsE-PE cells, suggesting gene amplification. Here, KRAS flanking genomic and transcriptomic regions were sequenced in CAsE-PE cells for insight into KRAS amplification. Comparison of DNA-Seq and RNA-Seq showed increased reads from background aligning to all KRAS exons in CAsE-PE cells, while a uniform DNA-Seq read distribution occurred in RWPE-1 cells with normal transcript expression. We searched for KRAS fusions in DNA and RNA sequencing data finding a portion of reads aligning to KRAS and viral sequence. After generation of cDNA from total RNA, short and long KRAS probes were generated to hybridize cDNA and KRAS enriched fragments were PacBio sequenced. More KRAS reads were captured from CAsE-PE cDNA versus RWPE-1 by each probe set. Only CAsE-PE cDNA showed KRAS viral fusion transcripts, primarily mapping to LTR and endogenous retrovirus sequences on either 5'- or 3'-ends of KRAS. Most KRAS viral fusion transcripts contained 4 to 6 exons but some PacBio sequences were in unusual orientations, suggesting viral insertions within the gene body. Additionally, conditioned media was extracted for potential retroviral particles. RNA-Seq of culture media isolates identified KRAS retroviral fusion transcripts in CAsE-PE media only. Truncated KRAS transcripts suggested multiple retroviral integration sites occurred within the KRAS gene producing KRAS retroviral fusions of various lengths. Findings suggest activation of endogenous retroviruses in arsenic carcinogenesis should be explored.

Arsenite malignantly transforms human prostate epithelial cells in vitro by gene amplification of mutated KRAS.

Inorganic arsenic is an environmental human carcinogen of several organs including the urinary tract. RWPE-1 cells are immortalized, non-tumorigenic, human prostate epithelia that become malignantly transformed into the CAsE-PE line after continuous in vitro exposure to 5µM arsenite over a period of months. For insight into in vitro arsenite transformation, we performed RNA-seq for differential gene expression and targeted sequencing of KRAS. We report >7,000 differentially expressed transcripts in CAsE-PE cells compared to RWPE-1 cells at >2-fold change, q<0.05 by RNA-seq. Notably, KRAS expression was highly elevated in CAsE-PE cells, with pathway analysis supporting increased cell proliferation, cell motility, survival and cancer pathways. Targeted DNA sequencing of KRAS revealed a mutant specific allelic imbalance, 'MASI', frequently found in primary clinical tumors. We found high expression of a mutated KRAS transcript carrying oncogenic mutations at codons 12 and 59 and many silent mutations, accompanied by lower expression of a wild-type allele. Parallel cultures of RWPE-1 cells retained a wild-type KRAS genotype. Copy number analysis and sequencing showed amplification of the mutant KRAS allele. KRAS is expressed as two splice variants, KRAS4a and KRAS4b, where variant 4b is more prevalent in normal cells compared to greater levels of variant 4a seen in tumor cells. 454 Roche sequencing measured KRAS variants in each cell type. We found KRAS4a as the predominant transcript variant in CAsE-PE cells compared to KRAS4b, the variant expressed primarily in RWPE-1 cells and in normal prostate, early passage, primary epithelial cells. Overall, gene expression data were consistent with KRAS-driven proliferation pathways found in spontaneous tumors and malignantly transformed cell lines. Arsenite is recognized as an important environmental carcinogen, but it is not a direct mutagen. Further investigations into this in vitro transformation model will focus on genomic events that cause arsenite-mediated mutation and overexpression of KRAS in CAsE-PE cells.

Opportunities and Challenges for Dietary Arsenic Intervention.

The diet is emerging as the dominant source of arsenic exposure for most of the U.S. population. Despite this, limited regulatory efforts have been aimed at mitigating exposure, and the role of diet in arsenic exposure and disease processes remains understudied. In this brief, we discuss the evidence linking dietary arsenic intake to human disease and discuss challenges associated with exposure characterization and efforts to quantify risks. In light of these challenges, and in recognition of the potential longer-term process of establishing regulation, we introduce a framework for shorter-term interventions that employs a field-to-plate food supply chain model to identify monitoring, intervention, and communication opportunities as part of a multisector, multiagency, science-informed, public health systems approach to mitigation of dietary arsenic exposure. Such an approach is dependent on coordination across commodity producers, the food industry, nongovernmental organizations, health professionals, researchers, and the regulatory community. https://doi.org/10.1289/EHP3997.

Pluripotent Stem Cells in Developmental Toxicity Testing: A Review of Methodological Advances.

Millions of children are born each year with a birth defect. Many of these defects are caused by environmental factors, although the underlying etiology is often unknown. In vivo mammalian models are frequently used to determine if a chemical poses a risk to the developing fetus. However, there are over 80 000 chemicals registered for use in the United States, many of which have undergone little safety testing, necessitating the need for higher-throughput methods to assess developmental toxicity. Pluripotent stem cells (PSCs) are an ideal in vitro model to investigate developmental toxicity as they possess the capacity to differentiate into nearly any cell type in the human body. Indeed, a burst of research has occurred in the field of stem cell toxicology over the past decade, which has resulted in numerous methodological advances that utilize both mouse and human PSCs, as well as cutting-edge technology in the fields of metabolomics, transcriptomics, transgenics, and high-throughput imaging. Here, we review the wide array of approaches used to detect developmental toxicants, suggest areas for further research, and highlight critical aspects of stem cell biology that should be considered when utilizing PSCs in developmental toxicity testing.

Arsenic Alters Exosome Quantity and Cargo to Mediate Stem Cell Recruitment Into a Cancer Stem Cell-Like Phenotype.

Inorganic arsenic is a human carcinogen that can target the prostate. Accumulating evidence suggests arsenic can disrupt stem cell (SC) dynamics during the carcinogenic process. Previous work demonstrated arsenic-transformed prostate epithelial (CAsE-PE) cells can recruit prostate SCs into rapidly acquiring a cancer SC (CSC) phenotype via the secretion of soluble factors. Exosomes are small, membrane-derived vesicles that contain lipids, RNA, and proteins, and actively contribute to cancer initiation and progression when taken up by target cells. Here we hypothesized that CAsE-PE cells are recruiting SCs to a CSC-like phenotype via exosomal signaling. CAsE-PE cells secreted 700% more exosomes than parental RWPE-1 cells. CAsE-PE exosomes were enriched with oncogenic factors, including oncogenes (KRAS, NRAS, VEFGA, MYB, and EGFR), inflammation-related (cyclooxygenase-2, interleukin 1B (IL1B), IL6, transforming growth factor-ß, and tumor necrosis factor-A), and apoptosis-related (CASP7, CASP9, and BCL2) transcripts, and oncogenesis-associated microRNAs. When compared with SCs cultured in exosome-depleted conditioned medium (CM), SCs cultured in CM containing CAsE-PE-derived exosomes showed increased (198%) matrix metalloproteinase activity and underwent an epithelial-to-mesenchymal transition in morphology, suggesting an exosome-mediated transformation. KRAS plays an important role in arsenic carcinogenesis. Although KRAS transcript (>24 000%) and protein (866%) levels were elevated in CAsE-PE exosomes, knock-down of KRAS in these cells only partially mitigated the CSC-like phenotype in cocultured SCs. Collectively, these results suggest arsenic impacts both exosomal quantity and cargo. Exosomal KRAS is only minimally involved in this recruitment, and additional factors (eg, cancer-associated miRNAs) likely also play a role. This work furthers our mechanistic understanding of how arsenic disrupts SC dynamics and influences the tumor microenvironment during carcinogenesis.

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.

Fluorescent proteins are an important tool that has become omnipresent in life sciences research. They are frequently used for localization of proteins and monitoring of cells [1,2]. Green fluorescent protein (GFP) was the first and has been the most used fluorescent protein. Enhanced GFP (eGFP) was optimized from wild-type GFP for increased fluorescence yield and improved expression in mammalian systems [3]. Many GFP-like fluorescent proteins have been discovered, optimized or created, such as the red fluorescent protein TagRFP [4]. Fluorescent proteins are expressed colorless and immature and, for eGFP, the conversion to the fluorescent form, mature, is known to produce one equivalent of hydrogen peroxide (H2O2) per molecule of chromophore [5,6]. Even though it has been proposed that this process is non-catalytic and generates nontoxic levels of H2O2 [6], this study investigates the role of fluorescent proteins in generating free radicals and inducing oxidative stress in biological systems. Immature eGFP and TagRFP catalytically generate the free radical superoxide anion (O2•-) and H2O2 in the presence of NADH. Generation of the free radical O2•- and H2O2 by eGFP in the presence of NADH affects the gene expression of cells. Many biological pathways are altered, such as a decrease in HIF1α stabilization and activity. The biological pathways altered by eGFP are known to be implicated in the pathophysiology of many diseases associated with oxidative stress; therefore, it is critical that such experiments using fluorescent proteins are validated with alternative methodologies and the results are carefully interpreted. Since cells inevitably experience oxidative stress when fluorescent proteins are expressed, the use of this tool for cell labeling and in vivo cell tracing also requires validation using alternative methodologies.

Haploinsufficiency of SIRT1 Enhances Glutamine Metabolism and Promotes Cancer Development.

SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, plays a vital role in the regulation of metabolism, stress responses, and genome stability. However, the role of SIRT1 in the multi-step process leading to transformation and/or tumorigenesis, as either a tumor suppressor or tumor promoter, is complex and may be dependent upon the context in which SIRT1 activity is altered, and the role of SIRT1 in tumor metabolism is unknown. Here, we demonstrate that SIRT1 dose-dependently regulates cellular glutamine metabolism and apoptosis, which in turn differentially impact cell proliferation and cancer development. Heterozygous deletion of Sirt1 induces c-Myc expression, enhancing glutamine metabolism and subsequent proliferation, autophagy, stress resistance, and cancer formation. In contrast, homozygous deletion of Sirt1 triggers cellular apoptotic pathways, increases cell death, diminishes autophagy, and reduces cancer formation. Consistent with the observed dose dependence in cells, intestine-specific Sirt1 heterozygous mice have enhanced intestinal tumor formation, whereas intestine-specific Sirt1 homozygous knockout mice have reduced development of colon cancer. Furthermore, SIRT1 reduction, but not deletion, is associated with human colorectal tumors, and colorectal cancer patients with low protein expression of SIRT1 have a poor prognosis. Taken together, our findings indicate that the dose-dependent regulation of tumor metabolism and possibly apoptosis by SIRT1 mechanistically contribute to the observed dual roles of SIRT1 in tumorigenesis. Our study highlights the importance of maintenance of a suitable SIRT1 dosage for metabolic and tissue homeostasis, which will have important implications in SIRT1-small-molecule-activator/inhibitor-based therapeutic strategies for cancers.