Acidity-mediated induction of FoxP3+ regulatory T cells.

Glucose limitation and increased lactic acid levels are consequences of the elevated glycolytic activity of tumor cells, and constitute a metabolic barrier for the function of tumor infiltrating effector immune cells. The immune-suppressive functions of regulatory T cells (Tregs) are unobstructed in lactic-acid rich environments. However, the impact of lactic acid on the induction of Tregs remains unknown. We observed increased TGFß-mediated induction of Forkhead box P3+ (FoxP3+ ) cells in the presence of extracellular lactic acid, in a glycolysis-independent, acidity-dependent manner. These CD4+ FoxP3+ cells expressed Treg-associated markers, including increased expression of CD39, and were capable of exerting suppressive functions. Corroborating these results in vivo, we observed that neutralizing the tumor pH by systemic administration of sodium bicarbonate (NaBi) decreased Treg abundance. We conclude that acidity augments Treg induction and propose that therapeutic targeting of acidity in the tumor microenvironment (TME) might reduce Treg-mediated immune suppression within tumors.

A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence.

In response to tenacious stress signals, such as the unscheduled activation of oncogenes, cells can mobilize tumour suppressor networks to avert the hazard of malignant transformation. A large body of evidence indicates that oncogene-induced senescence (OIS) acts as such a break, withdrawing cells from the proliferative pool almost irreversibly, thus crafting a vital pathophysiological mechanism that protects against cancer. Despite the widespread contribution of OIS to the cessation of tumorigenic expansion in animal models and humans, we have only just begun to define the underlying mechanism and identify key players. Although deregulation of metabolism is intimately linked to the proliferative capacity of cells, and senescent cells are thought to remain metabolically active, little has been investigated in detail about the role of cellular metabolism in OIS. Here we show, by metabolic profiling and functional perturbations, that the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) is a crucial mediator of senescence induced by BRAF(V600E), an oncogene commonly mutated in melanoma and other cancers. BRAF(V600E)-induced senescence was accompanied by simultaneous suppression of the PDH-inhibitory enzyme pyruvate dehydrogenase kinase 1 (PDK1) and induction of the PDH-activating enzyme pyruvate dehydrogenase phosphatase 2 (PDP2). The resulting combined activation of PDH enhanced the use of pyruvate in the tricarboxylic acid cycle, causing increased respiration and redox stress. Abrogation of OIS, a rate-limiting step towards oncogenic transformation, coincided with reversion of these processes. Further supporting a crucial role of PDH in OIS, enforced normalization of either PDK1 or PDP2 expression levels inhibited PDH and abrogated OIS, thereby licensing BRAF(V600E)-driven melanoma development. Finally, depletion of PDK1 eradicated melanoma subpopulations resistant to targeted BRAF inhibition, and caused regression of established melanomas. These results reveal a mechanistic relationship between OIS and a key metabolic signalling axis, which may be exploited therapeutically.

Phosphoproteome dynamics in onset and maintenance of oncogene-induced senescence.

Expression of the BRAF(V600E) oncoprotein is known to cause benign lesions, such as melanocytic nevi (moles). Despite the oncogenic function of mutant BRAF, these lesions are arrested by a cell-autonomous mechanism called oncogene-induced senescence. Infrequently, nevi can progress to malignant melanoma, through mechanisms that are incompletely understood. To gain more insight into this vital tumor-suppression mechanism, we performed a mass-spectrometry-based screening of the proteome and phosphoproteome in cycling and senescent cells and in cells with abrogated senescence. Proteome analysis of senescent cells revealed the up-regulation of established senescence biomarkers, including specific cytokines, but also several proteins not previously associated with senescence, including extracellular matrix-interacting. Using both general and targeted phosphopeptide enrichment by Ti(4+)-IMAC and phosphotyrosine antibody enrichment, we identified over 15,000 phosphorylation sites. Among the regulated phosphorylation sites we encountered components of the interleukin, BRAF/MAPK, and CDK-retinoblastoma pathways and several other factors. The extensive proteome and phosphoproteome dataset of BRAF(V600E)-expressing senescent cells provides molecular clues as to how oncogene-induced senescence is initiated, maintained, or evaded, serving as a comprehensive proteomic basis for functional validation.

Signal Transduction Reaction Monitoring Deciphers Site-Specific PI3K-mTOR/MAPK Pathway Dynamics in Oncogene-Induced Senescence.

We report a straightforward strategy to comprehensively monitor signal transduction pathway dynamics in mammalian systems. Combining targeted quantitative proteomics with highly selective phosphopeptide enrichment, we monitor, with great sensitivity, phosphorylation dynamics of the PI3K-mTOR and MAPK signaling networks. Our approach consists of a single enrichment step followed by a single targeted proteomics experiment, circumventing the need for labeling and immune purification while enabling analysis of selected phosphorylation nodes throughout signaling pathways. The need for such a comprehensive pathway analysis is illustrated by highlighting previously uncharacterized phosphorylation changes in oncogene-induced senescence, associated with diverse biological phenotypes and pharmacological intervention of the PI3K-mTOR pathway.

Two-way communication between the metabolic and cell cycle machineries: the molecular basis.

The relationship between cellular metabolism and the cell cycle machinery is by no means unidirectional. The ability of a cell to enter the cell cycle critically depends on the availability of metabolites. Conversely, the cell cycle machinery commits to regulating metabolic networks in order to support cell survival and proliferation. In this review, we will give an account of how the cell cycle machinery and metabolism are interconnected. Acquiring information on how communication takes place among metabolic signaling networks and the cell cycle controllers is crucial to increase our understanding of the deregulation thereof in disease, including cancer.

Near-genomewide RNAi screening for regulators of BRAF(V600E) -induced senescence identifies RASEF, a gene epigenetically silenced in melanoma.

The activation of oncogenes in primary cells blocks proliferation by inducing oncogene-induced senescence (OIS), a highly potent in vivo tumor-suppressing program. A prime example is mutant BRAF, which drives OIS in melanocytic nevi. Progression to melanoma occurs only in the context of additional alteration(s) like the suppression of PTEN, which abrogates OIS. Here, we performed a near-genomewide short hairpin (sh)RNA screen for novel OIS regulators and identified by next generation sequencing and functional validation seven genes. While all but one were upregulated in OIS, depletion of each of them abrogated BRAF(V) (600E) -induced arrest. With genome-wide DNA methylation analysis, we found one of these genes, RASEF, to be hypermethylated in primary cutaneous melanomas but not nevi. Bypass of OIS by depletion of RASEF was associated with suppression of several senescence biomarkers including senescence-associated (SA)-ß-galactosidase activity, interleukins, and tumor suppressor p15(INK) (4B) . Restoration of RASEF expression inhibited proliferation. These results illustrate the power of shRNA OIS bypass screens and identify a potential novel melanoma suppressor gene.