Chiara Gorrini

Modulation of oxidative stress as an anticancer strategy

The regulation of oxidative stress is an important factor in both tumour development and responses to anticancer therapies. Many signalling pathways that are linked to tumorigenesis can also regulate the metabolism of reactive oxygen species (ROS) through direct or indirect mechanisms. High ROS levels are generally detrimental to cells, and the redox status of cancer cells usually differs from that of normal cells. Because of metabolic and signalling aberrations, cancer cells exhibit elevated ROS levels. The observation that this is balanced by an increased antioxidant capacity suggests that high ROS levels may constitute a barrier to tumorigenesis. However, ROS can also promote tumour formation by inducing DNA mutations and pro-oncogenic signalling pathways. These contradictory effects have important implications for potential anticancer strategies that aim to modulate levels of ROS. In this Review, we address the controversial role of ROS in tumour development and in responses to anticancer therapies, and elaborate on the idea that targeting the antioxidant capacity of tumour cells can have a positive therapeutic impact.

Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly

The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.

Nuclear PTEN Controls DNA Repair and Sensitivity to Genotoxic Stress

PTEN Variations The product of the tumor suppressor gene phosphate and tensin homolog on chromosome ten (PTEN) is a lipid and protein phosphatase that regulates important cellular processes, including growth, survival, and metabolism (see the Perspective by Leslie and Brunton). Though PTEN is best known for effects on the phosphatidylnositol 3-kinase (PI3K) signaling pathway, the PTEN protein is also found in the nucleus. Bassi et al. (p. 395) found that PTENs presence in the nucleus was regulated in response to covalent modification of the protein by SUMOylation and phosphorylation. Cells lacking nuclear PTEN showed increased sensitivity to DNA damage and underwent cell death if the PI3K pathway was also inhibited. Hopkins et al. (p. 399, published online 6 June) discovered an alternative translation start site in human PTEN messenger RNA that allowed expression of a protein, PTEN-Long, with about 170 extra amino acids. The unusual enzyme was released from cells and then taken up into other cells. In a mouse tumor model, uptake of the enzyme inhibited the PI3K pathway and inhibited tumor growth. The phosphatase PTEN works as a lipid phosphatase in the cytoplasm and a protein phosphatase in the nucleus. [Also see Perspective by Leslie and Brunton] Loss of function of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene is associated with many human cancers. In the cytoplasm, PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K) signaling pathway. PTEN also accumulates in the nucleus, where its function remains poorly understood. We demonstrate that SUMOylation (SUMO, small ubiquitin-like modifier) of PTEN controls its nuclear localization. In cells exposed to genotoxic stress, SUMO-PTEN was rapidly excluded from the nucleus dependent on the protein kinase ataxia telangiectasia mutated (ATM). Cells lacking nuclear PTEN were hypersensitive to DNA damage, whereas PTEN-deficient cells were susceptible to killing by a combination of genotoxic stress and a small-molecule PI3K inhibitor both in vitro and in vivo. Our findings may have implications for individualized therapy for patients with PTEN-deficient tumors.

Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K–NRF2-regulated pathway

Significance Our establishment of a connection between the phosphatidylinositol 3-kinase (PI3K) and NRF2 pathways provides the basis for the tissue specificity of BRCA1-related cancers. Because BRCA1 is a vital component of the intracellular machinery maintaining genomic stability, BRCA1 functions as a major tumor suppressor in cells of all types. However, BRCA1-related cancers occur overwhelmingly in breasts and ovaries. Our work demonstrates that estrogen (E2) acts via the PI3K–AKT pathway to regulate NRF2 activation in BRCA1-deficient cells, resulting in the induction of antioxidant genes that protect the cell from reactive oxygen species-induced death. BRCA1-deficient cells are thus allowed to survive and may accumulate mutations, such as loss of PTEN, that perpetuate NRF2 activation. Our work supports the emerging clinical strategy of treating BRCA1-related cancers with PI3K inhibitors. Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1’s tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase–AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2.

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells

Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.

Glutathione Primes T Cell Metabolism for Inflammation

&NA; Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc‐deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin‐1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc‐dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T‐cell‐specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses. Graphical Abstract Figure. No caption available. HighlightsGlutathione (GSH) is not needed for early T cell activation but promotes T cell growthGSH supports mTOR and NFAT activity and drives glycolysis and glutaminolysisGclc‐derived GSH buffers ROS and regulates Myc‐dependent metabolic reprogrammingAblation of Gclc in T cells impairs inflammatory responses in vivo &NA; Upon activation, T cells adapt their metabolism to meet their increased bioenergetic and biosynthetic needs. Activated T cells produce ROS, which trigger the antioxidative GSH response to prevent cellular damage. Mak et al. report that the GSH pathway plays an unexpected role in metabolic integration during inflammatory T cell responses.

PTPN12 promotes resistance to oxidative stress and supports tumorigenesis by regulating FOXO signaling

It is well known that protein tyrosine phosphatases (PTPs) that become oxidized due to exposure to reactive oxygen species (ROS) undergo a conformational change and are inactivated. However, whether PTPs can actively regulate ROS levels in order to prevent PTP inhibition has yet to be investigated. Here, we demonstrate that PTP non-receptor type 12 (PTPN12) protects cells against aberrant ROS accumulation and death induced by oxidative stress. Murine embryonic fibroblasts (MEFs) deficient in PTPN12 underwent increased ROS-induced apoptosis under conditions of antioxidant depletion. Cells lacking PTPN12 also showed defective activation of FOXO1/3a, transcription factors required for the upregulation of several antioxidant genes. PTPN12-mediated regulation of ROS appeared to be mediated by phosphoinositide-dependent kinase-1 (PDK1), which was hyperstimulated in the absence of PTPN12. As tight regulation of ROS to sustain survival is a key feature of cancer cells, we examined PTPN12 levels in tumors from a cohort of breast cancer patients. Patients whose tumors showed high levels of PTPN12 transcripts had a significantly poorer prognosis. Analysis of tissues from patients with various breast cancer subtypes revealed that more triple-negative breast cancers, the most aggressive breast cancer subtype, showed high PTPN12 expression than any other subtype. Furthermore, both human breast cancer cells and mouse mammary epithelial tumor cells engineered to lack PTPN12 exhibited reduced tumorigenic and metastatic potential in vivo that correlated with their elevated ROS levels. The involvement of PTPN12 in the antioxidant response of breast cancer cells suggests that PTPN12 may represent a novel therapeutic target for this disease.

Acidic nuclear phosphoprotein 32kDa (ANP32)B-deficient mouse reveals a hierarchy of ANP32 importance in mammalian development

The highly conserved ANP32 proteins are proposed to function in a broad array of physiological activities through molecular mechanisms as diverse as phosphatase inhibition, chromatin regulation, caspase activation, and intracellular transport. On the basis of previous analyses of mice bearing targeted mutations of Anp32a or Anp32e, there has been speculation that all ANP32 proteins play redundant roles and are dispensable for normal development. However, more recent work has suggested that ANP32B may in fact have functions that are not shared by other ANP32 family members. Here we report that ANP32B expression is associated with a poor prognosis in human breast cancer, consistent with the increased levels of Anp32b mRNA present in proliferating wild-type (WT) murine embryonic fibroblasts and stimulated WT B and T lymphocytes. Moreover, we show that, contrary to previous assumptions, Anp32b is very important for murine embryogenesis. In a mixed genetic background, ANP32B-deficient mice displayed a partially penetrant perinatal lethality that became fully penetrant in a pure C57BL/6 background. Surviving ANP32B-deficient mice showed reduced viability due to variable defects in various organ systems. Study of compound mutants lacking ANP32A, ANP32B, and/or ANP32E revealed previously hidden roles for ANP32A in mouse development that became apparent only in the complete absence of ANP32B. Our data demonstrate a hierarchy of importance for the mammalian Anp32 genes, with Anp32b being the most critical for normal development.