Beyond Genetics: Metastasis as an Adaptive Response in Breast Cancer.

Metastatic disease represents the primary cause of breast cancer (BC) mortality, yet it is still one of the most enigmatic processes in the biology of this tumor. Metastatic progression includes distinct phases: invasion, intravasation, hematogenous dissemination, extravasation and seeding at distant sites, micro-metastasis formation and metastatic outgrowth. Whole-genome sequencing analyses of primary BC and metastases revealed that BC metastatization is a non-genetically selected trait, rather the result of transcriptional and metabolic adaptation to the unfavorable microenvironmental conditions which cancer cells are exposed to (e.g., hypoxia, low nutrients, endoplasmic reticulum stress and chemotherapy administration). In this regard, the latest multi-omics analyses unveiled intra-tumor phenotypic heterogeneity, which determines the polyclonal nature of breast tumors and constitutes a challenge for clinicians, correlating with patient poor prognosis. The present work reviews BC classification and epidemiology, focusing on the impact of metastatic disease on patient prognosis and survival, while describing general principles and current in vitro/in vivo models of the BC metastatic cascade. The authors address here both genetic and phenotypic intrinsic heterogeneity of breast tumors, reporting the latest studies that support the role of the latter in metastatic spreading. Finally, the review illustrates the mechanisms underlying adaptive stress responses during BC metastatic progression.

Hydrogen peroxide: a metabolic by-product or a common mediator of ageing signals?

The reactive oxygen species that are generated by mitochondrial respiration, including hydrogen peroxide (H2O2), are potent inducers of oxidative damage and mediators of ageing. It is not clear, however, whether oxidative stress is the result of a genetic programme or the by-product of physiological processes. Recent findings demonstrate that a fraction of mitochondrial H2O2, produced by a specialized enzyme as a signalling molecule in the pathway of apoptosis, induces intracellular oxidative stress and accelerates ageing. We propose that genes that control H2O2 production are selected determinants of lifespan.

S1P1 expression is controlled by the pro-oxidant activity of p66Shc and is impaired in B-CLL patients with unfavorable prognosis.

Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell-derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.

High-Fat Diet Promotes Acute Promyelocytic Leukemia through PPARδ-Enhanced Self-renewal of Preleukemic Progenitors.

Risk and outcome of acute promyelocytic leukemia (APL) are particularly worsened in obese-overweight individuals, but the underlying molecular mechanism is unknown. In established mouse APL models (Ctsg-PML::RARA), we confirmed that obesity induced by high-fat diet (HFD) enhances leukemogenesis by increasing penetrance and shortening latency, providing an ideal model to investigate obesity-induced molecular events in the preleukemic phase. Surprisingly, despite increasing DNA damage in hematopoietic stem cells (HSC), HFD only minimally increased mutational load, with no relevant impact on known cancer-driving genes. HFD expanded and enhanced self-renewal of hematopoietic progenitor cells (HPC), with concomitant reduction in long-term HSCs. Importantly, linoleic acid, abundant in HFD, fully recapitulates the effect of HFD on the self-renewal of PML::RARA HPCs through activation of peroxisome proliferator-activated receptor delta, a central regulator of fatty acid metabolism. Our findings inform dietary/pharmacologic interventions to counteract obesity-associated cancers and suggest that nongenetic factors play a key role. PREVENTION RELEVANCE: Our work informs interventions aimed at counteracting the cancer-promoting effect of obesity. On the basis of our study, individuals with a history of chronic obesity may still significantly reduce their risk by switching to a healthier lifestyle, a concept supported by evidence in solid tumors but not yet in hematologic malignancies. See related Spotlight, p. 47.

A Rare Subset of Primary Tumor Cells with Concomitant Hyperactivation of Extracellular Matrix Remodeling and dsRNA-IFN1 Signaling Metastasizes in Breast Cancer.

Metastatic breast cancer has a poor prognosis and is largely considered incurable. A better understanding of the molecular determinants of breast cancer metastasis could facilitate development of improved prevention and treatment strategies. We used lentiviral barcoding coupled to single-cell RNA sequencing to trace clonal and transcriptional evolution during breast cancer metastasis and showed that metastases derive from rare prometastatic clones that are underrepresented in primary tumors. Both low clonal fitness and high metastatic potential were independent of clonal origin. Differential expression and classification analyses revealed that the prometastatic phenotype was acquired by rare cells characterized by the concomitant hyperactivation of extracellular matrix remodeling and dsRNA-IFN signaling pathways. Notably, genetic silencing of key genes in these pathways (KCNQ1OT1 or IFI6, respectively) significantly impaired migration in vitro and metastasis in vivo, with marginal effects on cell proliferation and tumor growth. Gene expression signatures derived from the identified prometastatic genes predict metastatic progression in patients with breast cancer, independently of known prognostic factors. This study elucidates previously unknown mechanisms of breast cancer metastasis and provides prognostic predictors and therapeutic targets for metastasis prevention. SIGNIFICANCE: Transcriptional lineage tracing coupled with single-cell transcriptomics defined the transcriptional programs underlying metastatic progression in breast cancer, identifying prognostic signatures and prevention strategies.

Aberrant activation of p53/p66Shc-mInsc axis increases asymmetric divisions and attenuates proliferation of aged mammary stem cells.

Aging is accompanied by the progressive decline in tissue regenerative capacity and functions of resident stem cells (SCs). Underlying mechanisms, however, remain unclear. Here we show that, during chronological aging, self-renewing mitoses of mammary SCs (MaSCs) are preferentially asymmetric and that their progeny divides less frequently, leading to decreased number of MaSCs and reduced regenerative potential. Underlying mechanisms are investigated in the p66Shc-/- mouse, which exhibits several features of delayed aging, including reduced involution of the mammary gland (MG). p66Shc is a mitochondrial redox sensor that activates a specific p53 transcriptional program, in which the aging-associated p44 isoform of p53 plays a pivotal role. We report here that aged p66Shc-/- MaSCs show increased symmetric divisions, increased proliferation and increased regenerative potential, to an extent reminiscent of young wild-type (WT) MaSCs. Mechanistically, we demonstrate that p66Shc, together with p53: (i) accumulates in the aged MG, (ii) sustains expression of the cell polarity determinant mInscuteable and, concomitantly, (iii) down-regulates critical cell cycle genes (e.g.,: Cdk1 and Cyclin A). Accordingly, overexpression of p53/p44 increases asymmetric divisions and decreases proliferation of young WT MaSCs in a p66Shc-dependent manner and overexpression of mInsc restores WT-like levels of asymmetric divisions in aged p66Shc-/- MaSCs. Notably, deletion of p66Shc has negligible effects in young MaSCs and MG development. These results demonstrate that MG aging is due to aberrant activation of p66Shc, which induces p53/p44 signaling, leading to failure of symmetric divisions, decreased proliferation and reduced regenerative potential of MaSCs.

Decreased superoxide production in macrophages of long-lived p66Shc knock-out mice.

A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(-/-) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(-/-) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase Cdelta, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity.

Deletion of p66Shc longevity gene protects against experimental diabetic glomerulopathy by preventing diabetes-induced oxidative stress.

p66(Shc) regulates both steady-state and environmental stress-dependent reactive oxygen species (ROS) generation. Its deletion was shown to confer resistance to oxidative stress and protect mice from aging-associated vascular disease. This study was aimed at verifying the hypothesis that p66(Shc) deletion also protects from diabetic glomerulopathy by reducing oxidative stress. Streptozotocin-induced diabetic p66(Shc) knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas. Glomerular content of fibronectin and collagen IV was also lower in diabetic KO versus wild-type mice, whereas apoptosis was detected only in diabetic wild-type mice. Serum and renal tissue advanced glycation end products and plasma isoprostane 8-epi-prostaglandin F2alpha levels and activation of nuclear factor kappaB (NF-kappaB) were also lower in diabetic KO than in wild-type mice. Mesangial cells from KO mice grown under high-glucose conditions showed lower cell death rate, matrix production, ROS levels, and activation of NF-kappaB than those from wild-type mice. These data support a role for oxidative stress in the pathogenesis of diabetic glomerulopathy and indicate that p66(Shc) is involved in the molecular mechanism(s) underlying diabetes-induced oxidative stress and oxidant-dependent renal injury.

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells.

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.

Cooperation and selectivity of the two Grb2 binding sites of p52Shc in T-cell antigen receptor signaling to Ras family GTPases and Myc-dependent survival.

Shc proteins participate in a variety of processes regulating cell proliferation, survival and apoptosis. The two ubiquitously expressed isoforms, p52Shc/p46Shc, couple tyrosine kinase receptors to Ras by recruiting Grb2/Sos complexes to a membrane-proximal localization. Tyrosine residues 239/240 and 317 become phosphorylated following receptor engagement and, as such, form two Grb2 binding sites, which have been proposed to be differentially coupled to Myc-dependent survival and to fos-dependent proliferation, respectively. Here, we have addressed the individual function of YY239/240 and Y317 in T-cell antigen receptor (TCR) signaling. We show that p52Shc is phosphorylated on both YY239/240 and Y317 following TCR engagement. Mutation of either YY239/240 or Y317 results in impaired interaction with Grb2 and inhibition of Ras/MAP kinase activation and CD69 induction, supporting a role for both Grb2 binding sites in this function. Substitution of either YY239/240 or Y317 also results in a defective activation of Rac and the coupled stress kinases JNK and p38. Furthermore, mutation of Y317 or, to a larger extent, of YY239/240, results in increased activation-induced cell death, which in cells expressing the FF239/240 mutant is accompanied by impaired TCR-dependent c-myc transcription. The data underline a pleiotropic and nonredundant role of Shc, mediated by both YY239/240 and Y317, in T-cell activation and survival.

Apoptosis and aging: role of p66Shc redox protein.

p66Shc was the first mammalian gene whose mutation was demonstrated to increase resistance to oxidative stress and to prolong life span. Many hypotheses have been formulated to explain the biochemical and molecular basis of mammalian aging. Among them the free radical theory of aging, which was first proposed half a century ago by Harman, has received much attention by biomedical scientists. This theory proposed that, because of their high reactivity, reactive oxygen species (ROS) would lead to unavoidable and potentially deleterious by-products, and such an increasingly damaging process could be responsible for degenerative diseases and aging. Recent reports suggest an important role of p66Shc protein in the regulation of cellular responses to oxidative stress, apoptosis, and aging. In this review we discuss what has been discovered about p66Shc in the past 10 years and we focus particularly on its role in ROS regulation, which appears to be extremely promising to define mammalian aging processes.

P66SHC deletion improves fertility and progeric phenotype of late-generation TERC-deficient mice but not their short lifespan.

Oxidative stress and telomere attrition are considered the driving factors of aging. As oxidative damage to telomeric DNA favors the erosion of chromosome ends and, in turn, telomere shortening increases the sensitivity to pro-oxidants, these two factors may trigger a detrimental vicious cycle. To check whether limiting oxidative stress slows down telomere shortening and related progeria, we have investigated the effect of p66SHC deletion, which has been shown to reduce oxidative stress and mitochondrial apoptosis, on late-generation TERC (telomerase RNA component)-deficient mice having short telomeres and reduced lifespan. Double mutant (TERC(-/-) p66SHC(-/-) ) mice were generated, and their telomere length, fertility, and lifespan investigated in different generations. Results revealed that p66SHC deletion partially rescues sterility and weight loss, as well as organ atrophy, of TERC-deficient mice, but not their short lifespan and telomere erosion. Therefore, our data suggest that p66SHC-mediated oxidative stress and telomere shortening synergize in some tissues (including testes) to accelerate aging; however, early mortality of late-generation mice seems to be independent of any link between p66SHC-mediated oxidative stress and telomere attrition.

A p53-p66Shc signalling pathway controls intracellular redox status, levels of oxidation-damaged DNA and oxidative stress-induced apoptosis.

Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not influenced by p66Shc expression. In basal conditions, p66Shc-/- and p53-/- cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

Deletion of p66shc gene protects against age-related endothelial dysfunction.

BACKGROUND: Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction. The p66shc protein controls cellular responses to oxidative stress. Mice lacking p66shc (p66shc-/-) have increased resistance to ROS and a 30% prolonged life span. The present study investigates age-dependent changes of endothelial function in this model. METHODS AND RESULTS: Aortic rings from young and old p66shc-/- or wild-type (WT) mice were suspended for isometric tension recording. Nitric oxide (NO) release was measured by a porphyrinic microsensor. Expression of endothelial NO synthase (eNOS), inducible NOS (iNOS), superoxide dismutase, and nitrotyrosine-containing proteins was assessed by Western blotting. Nitrotyrosine residues were also identified by immunohistochemistry. Superoxide (O2-) production was determined by coelenterazine-enhanced chemiluminescence. Endothelium-dependent relaxation in response to acetylcholine was age-dependently impaired in WT mice but not in p66shc-/- mice. Accordingly, an age-related decline of NO release was found in WT but not in p66shc-/- mice. The expression of eNOS and manganese superoxide dismutase was not affected by aging either in WT or in p66shc-/- mice, whereas iNOS was upregulated only in old WT mice. It is interesting that old WT mice displayed a significant increase of O2- production as well as of nitrotyrosine expression compared with young animals. Such age-dependent changes were not found in p66shc-/- mice. CONCLUSIONS: We report that inactivation of the p66shc gene protects against age-dependent, ROS-mediated endothelial dysfunction. These findings suggest that the p66shc is part of a signal transduction pathway also relevant to endothelial integrity and may represent a novel target to prevent vascular aging.

Modelling the p53/p66Shc Aging Pathway in the Shortest Living Vertebrate Nothobranchius Furzeri.

Oxidative stress induced by reactive oxygen species (ROS) increases during lifespan and is involved in aging processes. The p66Shc adaptor protein is a master regulator of oxidative stress response in mammals. Ablation of p66Shc enhances oxidative stress resistance both in vitro and in vivo. Most importantly, it has been demonstrated that its deletion retards aging in mice. Recently, new insights in the molecular mechanisms involving p66Shc and the p53 tumor suppressor genes were given: a specific p66Shc/p53 transcriptional regulation pathway was uncovered as determinant in oxidative stress response and, likely, in aging. p53, in a p66Shc-dependent manner, negatively downregulates the expression of 200 genes which are involved in the G2/M transition of mitotic cell cycle and are downregulated during physiological aging. p66Shc modulates the response of p53 by activating a p53 isoform (p44/p53, also named Delta40p53). Based on these latest results, several developments are expected in the future, as the generation of animal models to study aging and the evaluation of the use of the p53/p66Shc target genes as biomarkers in aging related diseases. The aim of this review is to investigate the conservation of the p66Shc and p53 role in oxidative stress between fish and mammals. We propose to approach this study trough a new model organism, the annual fish Nothobranchius furzeri, that has been demonstrated to develop typical signs of aging, like in mammals, including senescence, neurodegeneration, metabolic disorders and cancer.

The influence of Shc proteins on life span in mice.

The signaling molecule p66Shc is often described as a longevity protein. This conclusion is based on a single life span study that used a small number of mice. The purpose of the present studies was to measure life span in a sufficient number of mice to determine if longevity is altered in mice with decreased Shc levels (ShcKO). Studies were completed at UC Davis and the European Institute of Oncology (EIO). At UC Davis, male C57BL/6J WT and ShcKO mice were fed 5% or 40% calorie-restricted (CR) diets. In the 5% CR group, there was no difference in survival curves between genotypes. There was also no difference between genotypes in prevalence of neoplasms or other measures of end-of-life pathology. At 40% calorie restriction group, 70th percentile survival was increased in ShcKO, while there were no differences between genotypes in median or subsequent life span measures. At EIO, there was no increase in life span in ShcKO male or female mice on C57BL/6J, 129Sv, or hybrid C57BL/6J-129Sv backgrounds. These studies indicate that p66Shc is not a longevity protein. However, additional studies are needed to determine the extent to which Shc proteins may influence the onset and severity of specific age-related diseases.

Deletion of p66Shc in mice increases the frequency of size-change mutations in the lacZ transgene.

Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2- to 24-month-old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size-change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X-ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lacZp66KO mice treated with H2 O2 . These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism.

p66Shc, mitochondria, and the generation of reactive oxygen species.

Reactive oxygen species (ROS), mainly originated from mitochondrial respiration, are critical inducers of oxidative damage and involved in tissue dysfunction. It is not clear, however, whether oxidative stress is the result of an active gene program or it is the by-product of physiological processes. Recent findings demonstrate that ROS are produced by mitochondria in a controlled way through specialized enzymes, including p66Shc, and take part in cellular process aimed to ensure adaptation and fitness. Therefore, genes generating specifically ROS are selected determinants of life span in response to different environmental conditions.

Oxidative stress activates a specific p53 transcriptional response that regulates cellular senescence and aging.

Oxidative stress is a determining factor of cellular senescence and aging and a potent inducer of the tumour-suppressor p53. Resistance to oxidative stress correlates with delayed aging in mammals, in the absence of accelerated tumorigenesis, suggesting inactivation of selected p53-downstream pathways. We investigated p53 regulation in mice carrying deletion of p66, a mutation that retards aging and confers cellular resistance and systemic resistance to oxidative stress. We identified a transcriptional network of ~200 genes that are repressed by p53 and encode for determinants of progression through mitosis or suppression of senescence. They are selectively down-regulated in cultured fibroblasts after oxidative stress, and, in vivo, in proliferating tissues and during physiological aging. Selectivity is imposed by p66 expression and activation of p44/p53 (also named Delta40p53), a p53 isoform that accelerates aging and prevents mitosis after protein damage. p66 deletion retards aging and increases longevity of p44/p53 transgenic mice. Thus, oxidative stress activates a specific p53 transcriptional response, mediated by p44/p53 and p66, which regulates cellular senescence and aging.