Showing papers by "Davide Ruggero published in 2018"

Development of a stress response therapy targeting aggressive prostate cancer.

Abstract: Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2–α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.

A PERK–miR-211 axis suppresses circadian regulators and protein synthesis to promote cancer cell survival

Abstract: The unfolded protein response (UPR) is a stress-activated signalling pathway that regulates cell proliferation, metabolism and survival. The circadian clock coordinates metabolism and signal transduction with light/dark cycles. We explore how UPR signalling interfaces with the circadian clock. UPR activation induces a 10 h phase shift in circadian oscillations through induction of miR-211, a PERK-inducible microRNA that transiently suppresses both Bmal1 and Clock, core circadian regulators. Molecular investigation reveals that miR-211 directly regulates Bmal1 and Clock via distinct mechanisms. Suppression of Bmal1 and Clock has the anticipated impact on expression of select circadian genes, but we also find that repression of Bmal1 is essential for UPR-dependent inhibition of protein synthesis and cell adaptation to stresses that disrupt endoplasmic reticulum homeostasis. Our data demonstrate that c-Myc-dependent activation of the UPR inhibits Bmal1 in Burkitt’s lymphoma, thereby suppressing both circadian oscillation and ongoing protein synthesis to facilitate tumour progression.

Revealing nascent proteomics in signaling pathways and cell differentiation.

Abstract: Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography-tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.

A wobbly road to drug resistance in melanoma: tRNA-modifying enzymes in translation reprogramming

Abstract: Alterations in transcript‐specific translation are emerging as a driver of cellular transformation and cancer etiology. A new study provides evidence for enhanced codon‐dependent translation of hypoxia‐inducible factor 1α in promoting glycolytic metabolism and drug resistance in melanoma cells. This specialized translation reprogramming relies, in part, on mTORC2‐mediated phosphorylation of enzymes modifying the wobble position of the transfer RNA anticodon.

Profiling the surface proteome identifies actionable biology for TSC1 mutant cells beyond mTORC1 signaling

Abstract: Loss of the TSC1/TSC2 complex leads to constitutively high mTORC1 signaling; however, pharmacological inhibition of mTORC1 in this setting produces a broad spectrum of clinical responses. We report herein several cell surface proteins upregulated by inactivation of TSC1 that present therapeutic alternatives or adjuvants to direct mTORC1 inhibition. A proteomics screen revealed that TSC1 loss most dramatically induced the expression of neprilysin (NEP/CD10) and aminopeptidase N (APN/CD13). The survival of TSC1 null human cancer cells was dependent on NEP expression, and TSC1 mutation sensitized cells to biochemical inhibition of APN. Remarkably, NEP and APN upregulation occurred via a TSC2- and mTORC1-independent mechanism; therefore, the antiproliferative effects of mTORC1 inhibition could be augmented by co-suppression of APN activity.