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Autophagy is an attractive therapeutic target in cancer. Successful autophagy-focused clinical intervention will require a detailed understanding of when and where autophagy is important during tumorigenesis. In this issue of Cancer Research, Khayati and colleagues use state-of-the-art genetically engineered mouse models to demonstrate that transient systemic inhibition of autophagy can irreversibly impair the growth of established lung tumors with a good tolerability in normal tissues, suggesting a therapeutic strategy for cancer treatment.See related article by Khayati et al., p. 4429

Ferroptosis is an iron-dependent form of regulated cell death induced by the lethal overload of lipid peroxides in cellular membranes. In recent years, modulating ferroptosis has gained attention as a potential therapeutic approach for tumor suppression. In the present study, retinol saturase (RETSAT) was identified as a significant ferroptosis mediator using a publicly accessible CRISPR/Cas9 screening dataset. RETSAT depletion protected tumor cells from lipid peroxidation and subsequent cell death triggered by various ferroptosis inducers. Furthermore, exogenous supplementation with retinoids, including retinol (the substrate of RETSAT) and its derivatives retinal and retinoic acid, also suppressed ferroptosis, whereas the product of RETSAT, 13, 14-dihydroretinol, failed to do so. As effective radical-trapping antioxidant, retinoids protected the lipid membrane from autoxidation and subsequent fragmentation, thus terminating the cascade of ferroptosis. Pseudotargeted lipidomic analysis identified an association between retinoid regulation of ferroptosis and lipid metabolism. Retinoic acid, but not 13, 14-dihydroretinoic acid, interacted with its nuclear receptor and activated transcription of SCD, which introduces the first double bond into saturated fatty acid and thus catalyzes the generation of monounsaturated fatty acid, a known ferroptosis suppressor. Therefore, RETSAT promotes ferroptosis by transforming retinol to 13, 14-dihydroretinol, thereby turning a strong anti-ferroptosis regulator into a relatively weak one.

Ferroptosis, a form of regulated cell death induced by excessive lipid peroxidation, has recently emerged as a key tumor suppression mechanism(1–4). Glutathione peroxidase 4 (GPX4)(5, 6) and ferroptosis suppressor protein 1 (FSP1)(7, 8) constitute two major ferroptosis defense systems. Here we show that treatment with GPX4 inhibitors in cancer cells results in an acute depletion of N-carbamoyl-L-aspartate (C-Asp), a pyrimidine biosynthesis intermediate, with a concomitant accumulation of uridine. Supplementation of dihydroorotate (DHO) or orotate (OA), the substrate and product of DHO dehydrogenase (DHODH), attenuates or potentiates ferroptosis induced by GPX4 inhibition, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4(low)). DHODH inactivation induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4(low) cancer cells, whereas DHODH inactivation synergizes with ferroptosis inducers to induce mitochondrial lipid peroxidation and ferroptosis in GPX4(high) cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independent of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane, through reducing ubiquinone (CoQ) to ubiquinol (CoQH(2)), a radical-trapping antioxidant with anti-ferroptosis activity. We further show that the DHODH inhibitor brequinar selectively suppresses GPX4(low) tumor growth through inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis inducing activity, synergistically induces ferroptosis and suppresses GPX4(high) tumor growth. Our results identify a DHODH-mediated ferroptosis defense mechanism in mitochondria and suggest a therapeutic strategy to target ferroptosis in cancer treatment.

Ferroptosis, a form of regulated cell death triggered by lipid peroxidation, was recently identified as an important mechanism in radiotherapy (RT)-mediated tumor suppression and radioresistance, although the exact genetic contexts in which to target ferroptosis in RT remains to be defined. p53 is the most commonly mutated gene in human cancers and a major effector to RT. Here, we identify ferroptosis as a critical mechanism to mediate p53 function in tumor radiosensitivity. Mechanistically, RT-mediated p53 activation antagonizes RT-induced SLC7A11 expression and represses glutathione synthesis, thereby promoting RT-induced lipid peroxidation and ferroptosis. p53 deficiency promotes radioresistance in cancer cells or tumors at least partly through SLC7A11-mediated ferroptosis inhibition. Ferroptosis inducers (FINs) that inhibit SLC7A11 exert significant radiosensitizing effects in tumor organoids and patient-derived xenografts with p53 mutation or deficiency. Finally, we show that RT-induced ferroptosis correlates with p53 activation and better clinical outcomes to RT in cancer patients. Together, our study uncovers a previously unappreciated role of ferroptosis in p53-mediated radiosensitization and suggest using FINs in combination with RT to treat p53-mutant cancers.

Ferroptosis, an iron-dependent form of regulated cell death driven by peroxidative damages of polyunsaturated-fatty-acid-containing phospholipids in cellular membranes, has recently been revealed to play an important role in radiotherapy-induced cell death and tumor suppression, and to mediate the synergy between radiotherapy and immunotherapy. In this review, we summarize known as well as putative mechanisms underlying the crosstalk between radiotherapy and ferroptosis, discuss the interactions between ferroptosis and other forms of regulated cell death induced by radiotherapy, and explore combination therapeutic strategies targeting ferroptosis in radiotherapy and immunotherapy. This review will provide important frameworks for future investigations of ferroptosis in cancer therapy.

ObjectiveGastro-oesophageal cancers (GEC) are resistant to therapy and lead to poor prognosis. The cancer stem cells (CSCs) and antiapoptotic pathways often confer therapy resistance. We sought to elucidate the antitumour action of a BCL-2 inhibitor, AT101 in GEC in vitro, in vivo and in a clinical trial.MethodsExtensive preclinical studies in vitro and in vivo were carried out to establish the mechanism action of AT101 on targeting CSCs and antiapoptotic proteins. A pilot clinical trial in patients with GEC was completed with AT-101 added to standard chemoradiation.ResultsOverexpression of BCL-2 and MCL-1 was noted in gastric cancer tissues (GC). AT-101 induced apoptosis, reduced proliferation and tumour sphere formation in MCL-1/BCL-2 high GC cells. Interestingly, AT101 dramatically downregulated genes (YAP-1/Sox9) that control CSCs in GEC cell lines regardless of BCL-2/MCL-1 expression. Addition of docetaxel to AT-101 amplified its antiproliferation and induced apoptosis effects. In vivo studies confirmed the combination of AT101 and docetaxel demonstrated stronger antitumour activity accompanied with significant decrease of CSCs biomarkers (YAP1/SOX9). In a pilot clinical trial, 13 patients with oesophageal cancer (EC) received AT101 orally concurrently with chemoradiation. We observed dramatic clinical complete responses and encouraging overall survival in these patients. Clinical specimen analyses revealed that AT-101 dramatically reduced the expression of CSCs genes in treated EC specimens indicating antitumour activity of AT101 relies more on its anti-CSCs activity.ConclusionsOur preclinical and clinical data suggest that AT-101 overcomes resistance by targeting CSCs pathways suggesting a novel mechanism of action of AT101 in patients with GEC.

The cystine/glutamate antiporter SLC7A11 (also commonly known as xCT) functions to import cystine for glutathione biosynthesis and antioxidant defense and is overexpressed in multiple human cancers. Recent studies revealed that SLC7A11 overexpression promotes tumor growth partly through suppressing ferroptosis, a form of regulated cell death induced by excessive lipid peroxidation. However, cancer cells with high expression of SLC7A11 (SLC7A11high) also have to endure the significant cost associated with SLC7A11-mediated metabolic reprogramming, leading to glucose- and glutamine-dependency in SLC7A11high cancer cells, which presents potential metabolic vulnerabilities for therapeutic targeting in SLC7A11high cancer. In this review, we summarize diverse regulatory mechanisms of SLC7A11 in cancer, discuss ferroptosis-dependent and -independent functions of SLC7A11 in promoting tumor development, explore the mechanistic basis of SLC7A11-induced nutrient dependency in cancer cells, and conceptualize therapeutic strategies to target SLC7A11 in cancer treatment. This review will provide the foundation for further understanding SLC7A11 in ferroptosis, nutrient dependency, and tumor biology and for developing novel effective cancer therapies.

Epigenetic regulation of gene transcription has been shown to coordinate with nutrient availability, yet the mechanisms underlying this coordination remain incompletely understood. Here, we show that glucose starvation suppresses histone 2A K119 monoubiquitination (H2Aub), a histone modification that correlates with gene repression. Glucose starvation suppressed H2Aub levels independently of energy stress–mediated AMP-activated protein kinase activation and possibly through NADPH depletion and subsequent inhibition of BMI1, an integral component of polycomb-repressive complex 1 (PRC1) that catalyzes H2Aub on chromatin. Integrated transcriptomic and epigenomic analyses linked glucose starvation–mediated H2Aub repression to the activation of genes involved in the endoplasmic reticulum (ER) stress response. We further showed that this epigenetic mechanism has a role in glucose starvation–induced cell death and that pharmacologic inhibition of glucose transporter 1 and PRC1 synergistically promoted ER stress and suppressed tumor growth in vivo. Together, these results reveal a hitherto unrecognized epigenetic mechanism coupling glucose availability to the ER stress response. Significance: These findings link glucose deprivation and H2A ubiquitination to regulation of the ER stress response in tumor growth and demonstrate pharmacologic susceptibility to inhibition of polycomb and glucose transporters.

SummaryUnderstanding metabolic dependencies and their therapeutic vulnerabilities is key to precision oncology. Malic enzyme 1 (ME1) is frequently overexpressed in cancers with protumorigenic effects. In contrast, consistent loss of ME1 expression in synovial sarcoma compared with other sarcomas indicates a unique ferroptosis liability for precision therapy in this form of cancer.See related article by Brashears et al., p. 3573

Ferroptosis, a form of regulated cell death caused by lipid peroxidation, was recently identified as a natural tumor suppression mechanism. Here, we show that ionizing radiation (IR) induces ferroptosis in cancer cells. Mechanistically, IR induces not only reactive oxygen species (ROS) but also the expression of ACSL4, a lipid metabolism enzyme required for ferroptosis, resulting in elevated lipid peroxidation and ferroptosis. ACSL4 ablation largely abolishes IR-induced ferroptosis and promotes radioresistance. IR also induces the expression of ferroptosis inhibitors, including SLC7A11 and GPX4, as an adaptive response. IR- or KEAP1 deficiency-induced SLC7A11 expression promotes radioresistance through inhibiting ferroptosis. Inactivating SLC7A11 or GPX4 with ferroptosis inducers (FINs) sensitizes radioresistant cancer cells and xenograft tumors to IR. Furthermore, radiotherapy induces ferroptosis in cancer patients, and increased ferroptosis correlates with better response and longer survival to radiotherapy in cancer patients. Our study reveals a previously unrecognized link between IR and ferroptosis and indicates that further exploration of the combination of radiotherapy and FINs in cancer treatment is warranted.

Summary Metabolic reprogramming in cancer cells can create metabolic liabilities. KEAP1-mutant lung cancer is refractory to most current therapies. Here we show that KEAP1 deficiency promotes glucose dependency in lung cancer cells, and KEAP1-mutant/deficient lung cancer cells are more vulnerable to glucose deprivation than their WT counterparts. Mechanistically, KEAP1 inactivation in lung cancer cells induces constitutive activation of NRF2 transcription factor and aberrant expression of NRF2 target cystine transporter SLC7A11; under glucose limitation, high cystine uptake in KEAP1-inactivated lung cancer cells stimulates toxic intracellular disulfide buildup, NADPH depletion, and cell death, which can be rescued by genetic ablation of NRF2-SLC7A11 axis or treatments inhibiting disulfide accumulation. Finally, we show that KEAP1-inactivated lung cancer cells or xenograft tumors are sensitive to glucose transporter inhibitor. Together, our results reveal that KEAP1 deficiency induces glucose dependency in lung cancer cells and uncover a therapeutically relevant metabolic liability., Graphical abstract, Highlights • Mutant KEAP1 exposes a metabolic liability provoked by high cystine uptake • KEAP1-NRF2-SLC7A11 axis drives glucose dependency • KEAP1 mutant NSCLC tumors are sensitive to GLUT inhibitor, Physiology; Cell biology; Cancer

Background: Ferroptosis is a form of regulated cell death caused by excessive accumulation of lipid peroxides on the cellular membrane. While impaired ferroptosis has been shown to contribute to tumor development, uncontrolled ferroptosis has been associated with neurodegeneration, acute kidney injury, and cardiovascular disease. To defend against ferroptosis, cells are equipped with several antioxidant systems. Glutathione peroxidase 4 (GPX4) is a major antioxidant enzyme that localizes in both the cytosol and the mitochondria and uses glutathione (GSH) as its cofactor to convert lipid hydroperoxides into non-toxic lipid alcohols. In addition, dihydroorotate dehydrogenase (DHODH) catalyzes the reduction of ubiquinone to ubiquinol in the inner mitochondrial membrane and inhibits mitochondrial lipid peroxidation independently of the GSH-GPX4 mechanism. Radiotherapy (RT) is one of the major therapies used to treat cancers. During RT, the radiolysis of water generates reactive oxygen species (ROS), subsequently leading to lipid peroxidation and ferroptosis. In our study, we aim to determine the role of DHODH in RT-induced ferroptosis and to evaluate the therapeutic potential of DHODH inhibitors as robust radiosensitizers in cancer treatment. Methods: Lipid peroxidation and mitochondrial lipid peroxidation were evaluated by flow cytometry. Ferroptosis marker PTGS2 expression levels were evaluated by real-time PCR. CRISPR-Cas9 system used to generate knock-out cancer cell lines. Patient-derived xenografts (PDX) co-treated with RT and DHODH inhibitors were employed to access the combination effect. Lipid peroxidation marker 4-HNE levels in tumors were evaluated by immunohistochemistry. Results: RT induced lipid peroxidation and PTGS2 expression in a variety of cancer cell lines. Genetic knockout of DHODH caused cells more sensitive to RT-induced ferroptosis. Similarly, pharmacological inhibition of DHODH sensitized cancer cells to RT-induced ferroptotic cell death both in vitro and in vivo. Mechanistically, RT induces ROS production in mitochondria, resulting in mitochondrial lipid peroxidation and ferroptosis. DHODH catalyzes the reduction of ubiquinone to ubiquinol, which acts as an antioxidant to inhibit lipid peroxidation in mitochondria. In general, DHODH inhibition significantly sensitizes cancer cells to RT-induced ferroptosis by decreasing ubiquinol levels in mitochondria. Conclusion: Our study suggests a model in which DHODH inhibition enhances RT-induced ferroptosis. DHODH inhibition significantly decreases the ubiquinol/ubiquinone ratio in mitochondria, thereby sensitizing cancer cells to RT-induced ferroptosis caused by mitochondrial lipid peroxidation. Together, our results demonstrate that DHODH inhibitors can be used as radiosensitizers through inducing ferroptosis and propose a novel combination strategy for cancer therapy. Citation Format: Chao Mao, Amber Horbath, Guang Lei, Boyi Gan. DHODH inhibition enhances radiotherapy-induced ferroptosis by promoting mitochondrial lipid peroxidation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5658.

Metabolic reprogramming in cancer cells can create metabolic liabilities which could be exploited therapeutically. Tumor suppressor KEAP1 is frequently mutated in lung cancer, and KEAP1-mutant lung cancers are refractory to most current therapies. Here we show that KEAP1 deficiency promotes glucose dependency in lung cancer cells, and KEAP1-mutant or -deficient lung cancer cells are more vulnerable to glucose deprivation than KEAP1-proficient lung cancer cells. Mechanistically, KEAP1 inactivation in lung cancer cells leads to constitutive activation of NRF2 transcription factor and aberrant expression of NRF2 target cystine transporter SLC7A11; under glucose limiting conditions, high cystine uptake in KEAP1-inactivated lung cancer cells results in toxic buildup of intracellular disulfide molecules, NADPH depletion, and subsequent cell death, which can be rescued by either genetic ablation of the NRF2-SLC7A11 signaling axis or treatments that prevent disulfide accumulation. Finally, we show that KEAP1-inactivated lung cancer cells or xenograft tumors are sensitive to glucose transporter (GLUT) inhibitor KL-11743. Together, our results reveal that KEAP1 deficiency induces glucose dependency in lung cancercells and uncover a metabolic liability that can be therapeutically targeted in KEAP1-mutant lung cancer.

MALAT1 has previously been described as a metastasis-promoting long noncoding RNA (lncRNA). We show here, however, that targeted inactivation of the Malat1 gene in a transgenic mouse model of breast cancer, without altering the expression of its adjacent genes, promotes lung metastasis, and that this phenotype can be reversed by genetic add-back of Malat1. Similarly, knockout of MALAT1 in human breast cancer cells induces their metastatic ability, which is reversed by re-expression of Malat1. Conversely, overexpression of Malat1 suppresses breast cancer metastasis in transgenic, xenograft, and syngeneic models. Mechanistically, the MALAT1 lncRNA binds and inactivates the prometastatic transcription factor TEAD, preventing TEAD from associating with its co-activator YAP and target gene promoters. Moreover, MALAT1 levels inversely correlate with breast cancer progression and metastatic ability. These findings demonstrate that MALAT1 is a metastasis-suppressing lncRNA rather than a metastasis promoter in breast cancer, calling for rectification of the model for this highly abundant and conserved lncRNA.

Ferroptosis, a form of regulated cell death triggered by lipid hydroperoxide accumulation, has an important role in a variety of diseases and pathological conditions, such as cancer. Targeting ferroptosis is emerging as a promising means of therapeutic intervention in cancer treatment. Polyunsaturated fatty acids, reactive oxygen species, and labile iron constitute the major underlying triggers for ferroptosis. Other regulators of ferroptosis have also been discovered recently, among them the mechanistic target of rapamycin complex 1 (mTORC1), a central controller of cell growth and metabolism. Inhibitors of mTORC1 have been used in treating diverse diseases, including cancer. In this review, we discuss recent findings linking mTORC1 to ferroptosis, dissect mechanisms underlying the establishment of mTORC1 as a key ferroptosis modulator, and highlight the potential of co-targeting mTORC1 and ferroptosis in cancer treatment. This review will provide valuable insights for future investigations of ferroptosis and mTORC1 in fundamental biology and cancer therapy.

Ferroptosis is a cell death mechanism triggered by lipid peroxidation. Our recent study linked cyst(e)ine availability with glutathione peroxidase 4 (GPX4) protein synthesis and ferroptosis mitigation via a Rag-mechanistic target of rapamycin complex 1 (mTORC1) axis, and proposed that co-targeting mTORC1 and ferroptosis is a promising strategy for cancer therapy.

Biguanides, including metformin (widely used in diabetes treatment) and phenformin, are AMP-activated protein kinase (AMPK) activators and potential drugs for cancer treatment. A more in-depth understanding of how cancer cells adapt to biguanide treatment may provide important therapeutic implications to achieve more effective and rational cancer therapies. NBR2 is a glucose starvation-induced long non-coding RNA (lncRNA) that interacts with AMPK and regulates AMPK activity upon glucose starvation. Here we show that phenformin treatment induces NBR2 expression, and NBR2 deficiency sensitizes cancer cells to phenformin-induced cell death. Surprisingly, unlike glucose starvation, phenformin does not induce NBR2 interaction with AMPK, and correspondingly, NBR2 deficiency does not affect phenformin-induced AMPK activation. We further reveal that NBR2 depletion attenuates phenformin-induced glucose transporter GLUT1 expression and glucose uptake. GLUT1 deficiency sensitizes cancer cells to phenformin-induced cell death, whereas GLUT1 restoration in NBR2 deficient cells rescues the increased cell death upon phenformin treatment. Together, the results of our study reveal that NBR2-GLUT1 axis may serve as an adaptive response in cancer cells to survive in response to phenformin treatment, and identify a novel mechanism coupling lncRNA to biguanide-mediated biology.

The invasive and metastatic properties of many human tumors have been associated with upregulation of the miRNA miR-10b, but its functional contributions in this setting have not been fully unraveled. Here, we report the generation of miR-10b–deficient mice, in which miR-10b is shown to be largely dispensable for normal development but critical to tumorigenesis. Loss of miR-10b delays oncogene-induced mammary tumorigenesis and suppresses epithelial–mesenchymal transition, intravasation, and metastasis in a mouse model of metastatic breast cancer. Among the target genes of miR-10b, the tumor suppressor genes Tbx5 and Pten and the metastasis suppressor gene Hoxd10 are significantly upregulated by miR-10b deletion. Mechanistically, miR-10b promotes breast cancer cell proliferation, migration, and invasion through inhibition of the expression of the transcription factor TBX5, leading to repression of the tumor suppressor genes DYRK1A and PTEN. In clinical specimens of breast cancer, the expression of TBX5, HOXD10, and DYRK1A correlates with relapse-free survival and overall survival outcomes in patients. Our results establish miR-10b as an oncomiR that drives metastasis, termed a metastamiR, and define the set of critical tumor suppressor mechanisms it overcomes to drive breast cancer progression. Cancer Res; 76(21); 6424–35. ©2016 AACR.

Introduction: Radiotherapy (RT) is widely used to treat cancers, but radioresistance remains a major factor in the failure of RT. RT generates reactive oxygen species (ROS) through the radiolysis of cellular water and the stimulation of oxidase, causing cellular damage. Ferroptosis is a regulated cell death induced by iron-dependent peroxidation of phospholipids containing polyunsaturated fatty acids (PUFA-PLs), which is morphologically and mechanistically distinct from apoptosis. There are several core regulators in ferroptosis pathway. SLC7A11 and GPX4 play a role in inhibiting ferroptosis, whereas ACSL4, as an essential lipid metabolism enzyme, promotes ferroptosis by regulating the biosynthesis of PUFA-PLs. Ferroptosis can be induced by either class 1 ferroptosis inducers (FINs) that inhibit SLC7A11 or class 2/3 FINs that inhibit or deplete GPX4. Our study aims to determine the role and mechanisms by which ferroptosis contributes to RT-induced cell death and tumor suppression and to evaluate therapeutic potential of FINs as robust radiosensitizers. Methods: Ferroptosis markers such as lipid peroxidation or PTGS2 expression was evaluated by flow cytometry, q-PCR or transmission electron microscopy (TEM). Mechanisms were analyzed by western blot and CRISPR-Cas9. Patient derived xenografts (PDX) co-treated with RT and FINs were employed to access the combination effectiveness. Samples were collected from patients before and after RT and ferroptosis marker 4-HNE was correlated with prognosis of patients. Results: RT induced lipid peroxidation and PTGS2 expression in a variety of cancer types and TEM revealed that cancer cells after RT exhibited shrunken mitochondria with enhanced membrane density, a morphologic feature of ferroptosis. Treatment with ferroptosis inhibitor ferrostatin-1 significantly restored cell survival that had been reduced by exposure to RT, suggesting ferroptosis represents an important part of the RT-induced cell death response. Mechanistically, RT up-regulated the expression of ACSL4, together with RT-generated ROS, to promote lipid peroxidation and ferroptosis. RT also induced the expression of ferroptosis negative regulators, including SLC7A11 and GPX4, leading to radioresistance. In preclinical model, ferroptosis inhibitor liproxstatin-1 partially abolished the RT-induced tumor suppression in H460 xenografts, and FINs (e.g. sulfasalazine) treatment exhibited a significant radiosensitization in A549 xenografts or radioresistant lung cancer patient-derived xenografts (PDXs). In clinical analysis, we revealed very weak 4-HNE levels in all tumor samples before RT, but RT increased 4-HNE levels moderately or strongly in all tumor samples. Importantly, the 4-HNE levels were positively correlated with RT response and disease-free survival in cancer patients. Conclusions: Our study reveals a previously unrecognized link between RT and ferroptosis, and demonstrates that FINs as a radiosensitizer reverse ferroptosis-mediated radioresistance, and proposes a novel combination therapeutic strategy for cancers, providing an effective approach to overcome radioresistance and facilitating us to initiate follow-up clinical trials to further explore the therapeutic potential of FINs combined with RT in cancer patients. Citation Format: Guang Lei, Yilei Zhang, Pranavi Koppula, Xiaoguang Liu, Steven Hsesheng Lin, Zhongxing Liao, Hui Wang, Boyi Gan. Ferroptosis plays an important role in the radiotherapy-induced tumor suppression and radioresistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2413.

Introduction Epigenetic regulation of gene transcription coordinates with nutrient availability, although the underlying mechanisms remain incompletely understood. Histone 2A monoubiquitination at lysine 119 (H2Aub), catalyzed mainly by its writer polycomb repressive complex 1 (PRC1), is a histone modification that has been associated with transcriptional repression. Initially, we found glucose withdrawal dramatically decreased H2Aub levels in multiple cancer cell lines. Next, we sought to study the role of H2Aub and H2Aub-associated transcriptional network in tumor cells while modulating glucose availability. Experimental design To determine the genes regulated by H2Aub upon glucose deprivation, H2Aub ChIP-seq and RNA-seq analyses were performed in UMRC6 kidney cancer cells with or without glucose. To identify the biological processes associated with the genes identified in our genome-wide analyses, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results We found that glucose starvation suppresses histone 2A K119 monoubiquitination (H2Aub), a histone modification that correlates with gene repression. Mechanistically, glucose starvation suppressed H2Aub levels independently of energy stress-mediated AMPK activation and likely acts through reactive oxygen species (ROS)-mediated inhibition of BMI1, an integral component of polycomb repressive complex 1 (PRC1) that catalyzes H2Aub on chromatin. Integrated transcriptomic and epigenomic analyses linked glucose starvation-mediated H2Aub repression to the activation of genes involved in the endoplasmic reticulum (ER) stress response. We further showed that this epigenetic mechanism has a role in glucose starvation-induced cell death. In addition, pharmacologic inhibition of glucose transporter 1 (GLUT1) and PRC1 synergistically promoted ER stress and suppressed tumor growth in vitro and in vivo. Conclusion Our study suggests a model in which glucose starvation inhibits PRC1 function potentially through glucose starvation-induced BMI phosphorylation and thereby decreases H2Aub levels, which leads to the de-repression of ER stress gene expression and contributes to the ER stress response and glucose starvation-induced cell death. Together, our results reveal a hitherto unrecognized epigenetic mechanism coupling glucose availability to the ER stress response. Citation Format: Yilei Zhang, Jiejun Shi, Zhenna Xiao, Guang Lei, Xiaoguang Liu, Hyemin Lee, Pranavi Koppula, Weijie Cheng, Chao Mao, Li Zhuang, Li Ma, Wei Li, Boyi Gan. H2A monoubiquitination links glucose availability to epigenetic regulation of the endoplasmic reticulum stress induced cell death in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2415.

The tumor suppressor BRCA1-associated protein 1 (BAP1) is a deubiquitinase that removes histone 2A ubiquitination. How BAP1 suppresses tumor development remains elusive. Our recent study identified the cystine transporter solute carrier family 7 member 11 (SLC7A11) as a critical BAP1 target, and showed that BAP1 promotes ferroptosis (a non-apoptotic cell death) through repressing SLC7A11 expression, resulting in tumor suppression.

Although cell-cycle arrest, senescence, and apoptosis are established mechanisms of tumor suppression, accumulating evidence reveals that ferroptosis, an iron-dependent, nonapoptotic form of cell death, represents a new regulatory pathway in suppressing tumor development. Ferroptosis is triggered by lipid peroxidation and is tightly regulated by SLC7A11, a key component of the cystine-glutamate antiporter. Although many studies demonstrate the importance of transcriptional regulation of SLC7A11 in ferroptotic responses, it remains largely unknown how the stability of SLC7A11 is controlled in human cancers. In this study, we utilized biochemial purification to identify the ubiquitin hydrolase OTUB1 as a key factor in modulating SLC7A11 stability. OTUB1 directly interacted with and stabilized SLC7A11; conversely, OTUB1 knockdown diminished SLC7A11 levels in cancer cells. OTUB1 was overexpressed in human cancers, and inactivation of OTUB1 destabilized SLC7A11 and led to growth suppression of tumor xenografts in mice, which was associated with reduced activation of ferroptosis. Notably, overexpression of the cancer stem cell marker CD44 enhanced the stability of SLC7A11 by promoting the interaction between SLC7A11 and OTUB1; depletion of CD44 partially abrogated this interaction. CD44 expression suppressed ferroptosis in cancer cells in an OTUB1-dependent manner. Together, these results show that OTUB1 plays an essential role in controlling the stability of SLC7A11 and the CD44-mediated effects on ferroptosis in human cancers. SIGNIFICANCE: This study identifies OTUB1 as a key regulator of ferroptosis and implicates it as a potential target in cancer therapy.

Depending on the circumstance, FOXO (Forkhead O) (FOXO1, FOXO3, and FOXO4) transcription factors activate the expression of markedly different sets of genes to produce different phenotypic effects. For example, distinct FOXO-regulated transcriptional programs stimulate cell death or enhance organism life span. To gain insight into how FOXOs select specific genes for regulation, we performed a screen for genes that modify FOXO activation of TRAIL, a death receptor ligand capable of inducing extrinsic apoptosis. We discovered that the bZIP transcriptional repressor NFIL3 (nuclear factor interleukin 3-regulated) hindered FOXO transcription factor access to chromatin at the TRAIL promoter by binding to nearby DNA and recruiting histone deacetylase-2 (HDAC2) to reduce histone acetylation. In the same manner, NFIL3 repressed expression of certain FOXO targets—e.g., FAS, GADD45α (growth arrest and DNA damage-inducible, α), and GADD45β—but not others. NFIL3, which we found to be overexpressed in different cancers, supported tumor cell survival largely through repression of TRAIL and antagonized hydrogen peroxide-induced cell death. Moreover, its expression in cancer was associated with lower patient survival. Therefore, NFIL3 alters cancer cell behavior and FOXO function by acting on chromatin to restrict the menu of FOXO target genes. Targeting of NFIL3 could be of therapeutic benefit for cancer patients.

NBR2 (neighbor of BRCA1 gene 2) is a non–protein coding gene that resides adjacent to tumor suppressor gene BRCA1, but its role in cancer biology has remained unknown. Our recent study showed that NBR2 encodes a long non-coding RNA and suppresses tumor development through regulation of adenosine monophosphate–activated protein kinase (AMPK) activation.

The roles of long non-coding RNAs in cancer metabolism remain largely unexplored. Here we identify FILNC1 (FoxO-induced long non-coding RNA 1) as an energy stress-induced long non-coding RNA by FoxO transcription factors. FILNC1 deficiency in renal cancer cells alleviates energy stress-induced apoptosis and markedly promotes renal tumor development. We show that FILNC1 deficiency leads to enhanced glucose uptake and lactate production through upregulation of c-Myc. Upon energy stress, FILNC1 interacts with AUF1, a c-Myc mRNA-binding protein, and sequesters AUF1 from binding c-Myc mRNA, leading to downregulation of c-Myc protein. FILNC1 is specifically expressed in kidney, and is downregulated in renal cell carcinoma; also, its low expression correlates with poor clinical outcomes in renal cell carcinoma. Together, our study not only identifies FILNC1 as a negative regulator of renal cancer with potential clinical value, but also reveals a regulatory mechanism by long non-coding RNAs to control energy metabolism and tumor development., FoxO are commonly down-regulated transcription factors and tumor suppressors in renal cell cancer (RCC). Here, the authors show that upon energy stress FoxOs induce the expression of the long non-coding RNA FILNC1, which inhibits survival of RCC by downregulating c-Myc and c-Myc-dependent metabolic rewiring.

SummaryTo assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm−/− mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1β, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1β or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.

SummaryA hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state, which contributes to its plasticity and therapeutic resistance. Here, integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/β-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells.

Cancer cells often upregulate nutrient transporters to fulfill their increased biosynthetic and bioenergetic needs, and to maintain redox homeostasis. One nutrient transporter frequently overexpressed in human cancers is the cystine/glutamate antiporter solute carrier family 7 member 11 (SLC7A11; also known as xCT). SLC7A11 promotes cystine uptake and glutathione biosynthesis, resulting in protection from oxidative stress and ferroptotic cell death. Recent studies have unexpectedly revealed that SLC7A11 also plays critical roles in glutamine metabolism and regulates the glucose and glutamine dependency of cancer cells. This review discusses the roles of SLC7A11 in regulating the antioxidant response and nutrient dependency of cancer cells, explores our current understanding of SLC7A11 regulation in cancer metabolism, and highlights key open questions for future studies in this emerging research area. A deeper understanding of SLC7A11 in cancer metabolism may identify new therapeutic opportunities to target this important amino acid transporter for cancer treatment.

The PI3K–AKT pathway is hyperactivated in many human cancers, and several drugs to inhibit this pathway, including the PI3K/mTOR dual inhibitor NVP-BEZ235, are currently being tested in various preclinical and clinical trials. It has been shown that pharmacologic inhibition of the PI3K–AKT pathway results in feedback activation of other oncogenic signaling pathways, which likely will limit the clinical utilization of these inhibitors in cancer treatment. However, the underlying mechanisms of such feedback regulation remain incompletely understood. The PI3K–AKT pathway is a validated therapeutic target in renal cell carcinoma (RCC). Here, we show that FoxO transcription factors serve to promote AKT phosphorylation at Ser473 in response to NVP-BEZ235 treatment in renal cancer cells. Inactivation of FoxO attenuated NVP-BEZ235–induced AKT Ser473 phosphorylation and rendered renal cancer cells more susceptible to NVP-BEZ235–mediated cell growth suppression in vitro and tumor shrinkage in vivo. Mechanistically, we showed that FoxOs upregulated the expression of Rictor, an essential component of MTOR complex 2, in response to NVP-BEZ235 treatment and revealed that Rictor is a key downstream target of FoxOs in NVP-BEZ235–mediated feedback regulation. Finally, we show that FoxOs similarly modulate the feedback response on AKT Ser473 phosphorylation and renal tumor growth by other phosphoinositide 3-kinase (PI3K) or AKT inhibitor treatment. Together, our study reveals a novel mechanism of PI3K–AKT inhibition-mediated feedback regulation and may identify FoxO as a novel biomarker to stratify patients with RCC for PI3K or AKT inhibitor treatment, or a novel therapeutic target to synergize with PI3K–AKT inhibition in RCC treatment. Cancer Res; 74(6); 1682–93. ©2014 AACR.

Normal cells possess adaptive mechanisms to couple energy availability with cell growth (cell size increase) and survival, and imbalances are associated with major diseases such as cancer. Inactivation of critical regulators involved in energy stress response, including adenosine monophosphate-activated protein kinase (AMPK), liver kinase B1 (LKB1), tuberous sclerosis complex 1 (TSC1) and tuberous sclerosis complex 2 (TSC2), leads to uncontrolled cell growth yet increased apoptosis under energy stress. These energy stress regulators are also important in tumor suppression and metabolism. Here, we show that forkhead box O (FoxO) transcription factor, a central regulator of tumor suppression and metabolism, plays a unique role in energy stress response. FoxOs inhibit the mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cell growth, under energy stress, and inactivation of FoxOs alleviates energy stress-mediated mTORC1 repression. Surprisingly, unlike AMPK-, Lkb1- or Tsc1/2-deficient cells, FoxO-deficient cells exhibit decreased apoptosis under energy stress. FoxOs operate to inhibit mTORC1 signaling and cell survival independent of AMPK and TSC. Integrated transcriptomic and functional analyses identified BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)-a negative regulator of both Rheb and Bcl2 prosurvival family members-as a key downstream target of FoxOs to inhibit mTORC1 function and promote apoptosis in response to energy stress. We show that p38β, but not AMPK, is likely to function upstream of FoxO-BNIP3 to mediate energy stress response. Finally, we reveal that low expression of FoxO or BNIP3 correlates with poor clinical outcomes in renal cancer patients. Together, our study uncovers a novel signaling circuit functioning to mediate cellular energy responses to control cell growth and survival. These findings also have important implications to human cancers.

Mammalian target of rapamycin (mTOR) is an important, yet poorly understood integrative kinase that regulates immune cell function. mTOR functions in 2 independent complexes: mTOR complex (mTORC) 1 and 2. The immunosuppressant rapamycin (RAPA) inhibits mTORC1 but not mTORC2 and causes a paradoxical reduction in anti-inflammatory interleukin (IL) 10 and B7-homolog 1 (B7-H1) expression by dendritic cells (DCs). Using catalytic mTOR inhibitors and DCs lacking mTORC2, we show that restraint of signal transducer and activator of transcription 3-mediated IL-10 and B7-H1 expression during DC maturation involves a RAPA-insensitive and mTORC2-independent mTOR mechanism. Relatedly, catalytic mTOR inhibition promotes B7-H1-dependent and IL-1β-dependent DC induction of regulatory T cells (Tregs). Thus, we define an immunoregulatory pathway in which RAPA-sensitive mTORC1 in DCs promotes effector T-cell expansion and RAPA-insensitive mTORC1 restrains T(reg) induction. These findings identify the first known RAPA-insensitive mTOR pathway that is not mediated solely by mTORC2 and have implications for the use of catalytic mTOR inhibitors in inflammatory disease settings.

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context-dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible KrasG12D-driven PDAC mouse model establishes that advanced PDAC remain strictly dependent on KrasG12D expression. Transcriptome and metabolomic analysis indicate that KrasG12D serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that KrasG12D drives glycolysis intermediates into the non-oxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC. Citation Format: Haoqiang Ying, Hailei Zhang, Jonathan L. Coloff, Haiyan Yan, Wei Wang, Shujuan Chen, Andrea Viale, Hongwu Zheng, Ji-hye Paik, Carol Lim, Alexander R. Guimaraes, Alec C. Kimmelman, Eric S. Martin, Jeffery Chang, Aram Hezel, Samuel R. Perry, Jian Hu, Boyi Gan, Yonghong Xiao, John M. Asara, Ralph Weissleder, Y. Alan Wang, Costas A. Lyssiotis, Lynda Chin, Lewis C. Cantley, Ronald A. DePinho, Sujun Hua, Gerald C. Chu, Eliot Fletcher-Sananikone, Jason W. Locasale, Jaekyoung Son. Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A101.

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.

Autophagy is a conserved cellular process for bulk degradation of intracellular protein and organelles in lysosomes. In contrast to elegant studies of beclin1 using mouse models and cultured cells demonstrating a tumor suppression function for autophagy, knockout of other essential autophagy proteins such as ATG5, ATG7, or FIP200 (FAK family-interacting protein of 200 kDa) in various tissues did not lead to malignant tumor development in vivo. Here, we report that inhibition of autophagy by FIP200 ablation suppresses mammary tumor initiation and progression in a mouse model of breast cancer driven by the PyMT oncogene. Deletion of FIP200 resulted in multiple autophagy defects including accumulation of ubiquitinated protein aggregates and p62/SQSTM1, deficient LC3 conversion, and increased number of mitochondria with abnormal morphology in tumor cells. FIP200 deletion did not affect apoptosis of mammary tumor cells or Ras-transformed mouse embryonic fibroblasts (MEFs), but significantly reduced their proliferation in both systems. We also observed a reduced glycolysis and cyclin D1 expression in FIP200-null mammary tumor cells and transformed MEFs. In addition, gene profiling studies revealed significantly elevated expression of interferon (IFN)-responsive genes in the early tumors of FIP200 conditional knockout mice, which was accompanied by increased infiltration of effector T cells in the tumor microenvironment triggered by an increased production of chemokines including CXCL10 in FIP200-null tumor cells. Together, these data provide strong evidence for a protumorigenesis role of autophagy in oncogene-induced tumors in vivo and suggest FIP200 as a potential target for cancer therapy.

SummarymTORC1 is a validated therapeutic target for renal cell carcinoma (RCC). Here, analysis of Tsc1-deficient (mTORC1 hyperactivation) mice uncovered a FoxO-dependent negative feedback circuit constraining mTORC1-mediated renal tumorigenesis. We document robust FoxO activation in Tsc1-deficient benign polycystic kidneys and FoxO extinction on progression to murine renal tumors; murine renal tumor progression on genetic deletion of both Tsc1 and FoxOs; and downregulated FoxO expression in most human renal clear cell and papillary carcinomas, yet continued expression in less aggressive RCCs and benign renal tumor subtypes. Mechanistically, integrated analyses revealed that FoxO-mediated block operates via suppression of Myc through upregulation of the Myc antagonists, Mxi1-SRα and mir-145, establishing a FoxO-Mxi1-SRα/mir-145 axis as a major progression block in renal tumor development.

The stringent regulation of hematopoietic stem cell (HSC) quiescence versus cell cycle progression is essential for the preservation of a pool of long-term self-renewing cells and vital for sustaining an adequate supply of all blood lineages throughout life. Cell growth, the process that is mass increase, serves as a trigger for cell cycle progression and is regulated predominantly by mammalian target of rapamycin complex 1 (mTORC1) signaling. Emerging data from various mice models show deletion of several mTORC1 negative regulators, including PTEN, TSC1, PML and Fbxw7 result in similar HSC phenotypes characterized as HSC hyper-proliferation and subsequent exhaustion, and defective repopulating potential. Further pharmacological approaches show that PTEN, TSC1 and PML regulate HSC maintenance through mTORC1. mTORC1-mediated cell growth regulatory circuits thus plays a critical role in the regulation of HSC quiescence.

The PI3K-AKT pathway is hyperactivated in many human cancers, and several drugs to inhibit this pathway are currently being tested in various pre-clinical and clinical trials. It has been shown that pharmacological inhibition of the PI3K-AKT pathway results in feedback activation of other oncogenic signaling pathways, which likely will limit the clinical utilization of these inhibitors in cancer treatment. However, the underlying mechanisms of such feedback regulation remain incompletely understood. The PI3K-AKT pathway is a validated therapeutic target in renal cell carcinoma (RCC). Here we show that FoxO transcription factors serve to promote AKT phosphorylation at Ser473 in response to the treatment of NVP-BEZ235, a PI3K/mTOR dual inhibitor, in renal cancer cells. Inactivation of FoxO attenuated NVP-BEZ235-induced AKT Ser473 phosphorylation, and rendered renal cancer cells more susceptible to NVP-BEZ235-mediated cell growth suppression in vitro and tumor shrinkage in vivo. Mechanistically, we showed that FoxOs upregulated the expression of Rictor, an essential component of mammalian target of rapamycin complex 2 (mTORC2), in response to NVP-BEZ235 treatment, and revealed that Rictor is a key downstream target of FoxOs in NVP-BEZ235-mediated feedback regulation. We also show that FoxOs similarly modulate the feedback response on AKT Ser473 phosphorylation and renal tumor growth by the treatment of other PI3K or AKT inhibitors, such as MK-2206. Together, our study reveals a novel mechanism of PI3K-AKT inhibition-mediated feedback regulation. Importantly, a recent RCC clinical trial with MK-2206 revealed a dichotomous response in renal cancer patients: a subset of patients had dramatic tumor reduction, while another subset of RCC patients showed no decrease in tumor growth rate, and had a very short progression free survival. We will discuss our work to determine the expression/activation status of the FoxO-Rictor-AKT signaling axis in renal cancer patients receiving AKT inhibitor treatment, which may identify FoxO as a novel biomarker to stratify RCC patients for PI3K or AKT inhibitor treatment. Citation Format: Boyi Gan. The roles of FoxO transcription factors in mediating AKT activation and renal tumor growth in response to pharmacological inhibition of the PI3K-AKT pathway. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B06.

Tuberous sclerosis complex 1 (TSC1) and TSC2 tumor suppressor proteins have been shown to negatively regulate cell growth through inhibition of the mammalian target of rapamycin (mTOR) pathway. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a critical role in integrin signaling. Here we identify a novel interaction between FAK and TSC2 and show that TSC2 is phosphorylated by FAK. Furthermore, we show that overexpression of FAK kinase dead mutant inhibits the phosphorylation of ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein-1, two key mTOR (mammalian target of rapamycin) downstream targets, and negatively regulates the cell size and that FAK regulation of S6K phosphorylation is through TSC2. Finally, we provide data that FAK plays a positive role in cell adhesion-induced S6K phosphorylation, whereas TSC2 is required for cell suspension-induced S6K inactivation. Together, these results suggest that FAK might regulate S6K activation and cell size through its interaction with and phosphorylation of TSC2 and also provide a previously unappreciated role of TSC2 in the regulation of mTOR signaling by cell adhesion.

FIP200 is a novel protein inhibitor for focal adhesion kinase (FAK), which binds to FAK directly and inhibits its kinase activity and associated cellular functions, such as cell adhesion, spreading, and motility in fibroblasts. Here we show that FIP200 inhibits G1-S phase progression, proliferation, and clonogenic survival in human breast cancer cells. Consistent with the G1 arrest induced by FIP200, we found that FIP200 increased p21 and decreased cyclin D1 protein levels in breast cancer cells. In addition, FIP200 significantly induced p21 promoter activity in MCF-7 cells and this response was abolished upon deletion of p53 binding sites within p21 promoter. Furthermore, we found that FIP200 could interact with exogenous and endogenous p53 protein and significantly increase its half-life compared with the control cells. We also found that the NH2-terminal 154 residues of FIP200 were sufficient to mediate p53 interaction and G1 arrest in cells. The increase in p53 half-life correlated with the increased phosphorylation at Ser15 and decreased proteasomal degradation via ubiquitin and Hdm2-independent mechanism. Stabilization of p53 by FIP200 could be partially reversed by NQO1 inhibitor, dicoumarol. In contrast to p53, FIP200 decreased cyclin D1 protein half-life by promoting proteasome-dependent degradation of cyclin D1. In summary, our results suggest that FIP200 increases p21 protein levels via stabilization of its upstream regulator p53 and decreases cyclin D1 protein by promoting its degradation. Both effects are critical for FIP200-induced G1 arrest and may contribute to the putative antitumor activities of FIP200 in breast cancer.

SummaryTo determine the role of telomere dysfunction and telomerase reactivation in generating pro-oncogenic genomic events and in carcinoma progression, an inducible telomerase reverse transcriptase (mTert) allele was crossed onto a prostate cancer-prone mouse model null for Pten and p53 tumor suppressors. Constitutive telomerase deficiency and associated telomere dysfunction constrained cancer progression. In contrast, telomerase reactivation in the setting of telomere dysfunction alleviated intratumoral DNA-damage signaling and generated aggressive cancers with rearranged genomes and new tumor biological properties (bone metastases). Comparative oncogenomic analysis revealed numerous recurrent amplifications and deletions of relevance to human prostate cancer. Murine tumors show enrichment of the TGF-β/SMAD4 network, and genetic validation studies confirmed the cooperative roles of Pten, p53, and Smad4 deficiencies in prostate cancer progression, including skeletal metastases. Thus, telomerase reactivation in tumor cells experiencing telomere dysfunction enables full malignant progression and provides a mechanism for acquisition of cancer-relevant genomic events endowing new tumor biological capabilities.