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Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia

Garcia-Bermudez, Javier; Badgley, Michael A; Prasad, Sheela; Baudrier, Lou; Liu, Yuyang; La, Konnor; Soula, Mariluz; Williams, Robert T; Yamaguchi, Norihiro; Hwang, Rosa F; Taylor, Laura J; de Stanchina, Elisa; Rostandy, Bety; Alwaseem, Hanan; Molina, Henrik; Bar-Sagi, Dafna; Birsoy, Kıvanç
Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.
PMID: 35726024
ISSN: 2522-5812
CID: 5278652

Wild-type h- and N-ras promote mutant k-ras-driven tumorigenesis by modulating the DNA damage response

Grabocka, Elda; Pylayeva-Gupta, Yuliya; Jones, Mathew J K; Lubkov, Veronica; Yemanaberhan, Eyoel; Taylor, Laura; Jeng, Hao Hsuan; Bar-Sagi, Dafna
Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anticancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here, we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways and, consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo.
PMID: 24525237
ISSN: 1535-6108
CID: 811132

EZH2 couples pancreatic regeneration to neoplastic progression

Mallen-St Clair, Jon; Soydaner-Azeloglu, Rengin; Lee, Kyoung Eun; Taylor, Laura; Livanos, Alexandra; Pylayeva-Gupta, Yuliya; Miller, George; Margueron, Raphael; Reinberg, Danny; Bar-Sagi, Dafna
Although the polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) is well recognized for its role as a key regulator of cell differentiation, its involvement in tissue regeneration is largely unknown. Here we show that EZH2 is up-regulated following cerulein-induced pancreatic injury and is required for tissue repair by promoting the regenerative proliferation of progenitor cells. Loss of EZH2 results in impaired pancreatic regeneration and accelerates KRas(G12D)-driven neoplasia. Our findings implicate EZH2 in constraining neoplastic progression through homeostatic mechanisms that control pancreatic regeneration and provide insights into the documented link between chronic pancreatic injury and an increased risk for pancreatic cancer.
PMID: 22391448
ISSN: 0890-9369
CID: 159852

Sos-mediated cross-activation of wild-type Ras by oncogenic Ras is essential for tumorigenesis

Jeng, Hao-Hsuan; Taylor, Laura J; Bar-Sagi, Dafna
Mammalian cells contain three closely related ras genes, H-ras, K-ras and N-ras. Although in a given tumour type, oncogenic mutations are selectively observed in only one of the ras genes, the acquisition of the transformed phenotype has been shown to require the contribution of the normal products of the other ras genes. Here we demonstrate that oncogenic K-Ras promotes the activation of wild-type H- and N-Ras. This activation is mediated by oncogenic K-Ras-dependent allosteric stimulation of Sos and confers a growth advantage to oncogenic K-Ras harbouring cancer cells. These findings underscore the complementary functions of oncogenic and wild-type Ras in tumour cells and identify a potential new targeting strategy for Ras-driven tumours.
PMID: 23132018
ISSN: 2041-1723
CID: 202202

Perturbation of cytoskeleton dynamics by the opposing effects of Rac1 and Rac1b

Nimnual AS; Taylor LJ; Nyako M; Jeng HH; Bar-Sagi D
Rac1, a ubiquitously expressed member of the Rho GTPase family, plays a pivotal role in the regulation of multiple cellular processes including cytoskeleton reorganization, cell growth, differentiation and motility. Here we show that the tumor-specific splice variant of Rac1, Rac1b, negatively regulates Rac1 activity. The expression of Rac1b in HeLa cells interferes with Rac1 activation by PDGF, leads to a reduction in membrane-bound Rac1 and promotes an increase in Rho activity. The antagonistic relationship between Rac1 and Rac1b perturbs the regulatory circuitry that controls actin cytoskeleton dynamics thereby leading to tumor-linked alterations in cell morphology and motility
PMID: 21686260
ISSN: 2154-1256
CID: 138351

Feedback regulation of Ras signaling by Rabex-5-mediated ubiquitination

Xu, Lizhong; Lubkov, Veronica; Taylor, Laura J; Bar-Sagi, Dafna
Ras proteins play a central role in transducing signals that control cell proliferation, differentiation, motility, and survival. The location-specific signaling activity of Ras has been previously shown to be regulated by ubiquitination [1]. However, the molecular machinery that controls Ras ubiquitination has not been defined. Here we demonstrate through biochemical and functional analyses that Rabex-5 (also known as RabGEF1) [2, 3] functions as an E3 ligase for Ras. Rabex-5-mediated Ras ubiquitination promotes Ras endosomal localization and leads to the suppression of ERK activation. Moreover, the Ras effector RIN1 [4, 5] is required for Rabex-5-dependent Ras ubiquitination, suggesting a feedback mechanism by which Ras activation can be coupled to ubiquitination. These findings define new elements in the regulatory circuitry that link Ras compartmentalization to signaling output
PMID: 20655225
ISSN: 1879-0445
CID: 138182

Spatial Regulation of EGFR Signaling by Sprouty2

Kim, Hong Joo; Taylor, Laura J; Bar-Sagi, Dafna
Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates multiple signal-transduction pathways as well as trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Although there is growing awareness that endocytic transport can play a direct role in signal specification, relatively little is known about the molecular mechanisms underlying this link. Here we show that human Sprouty 2 (hSpry2), a protein that has been implicated in the negative regulation of receptor tyrosine kinase (RTK) signaling [1], interferes with the trafficking of activated EGFR specifically at the step of progression from early to late endosomes. This effect is mediated by the binding of hSpry2 to the endocytic regulatory protein, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and leads to a block in intracellular signal propagation. These observations suggest that EGFR signaling is controlled by a novel mechanism involving trafficking-dependent alterations in receptor compartmentalization
PMID: 17320394
ISSN: 0960-9822
CID: 70959

Redox-dependent downregulation of Rho by Rac

Nimnual, Anjaruwee S; Taylor, Laura J; Bar-Sagi, Dafna
Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases
PMID: 12598902
ISSN: 1465-7392
CID: 62933

Induction of the cellular E2F-1 promoter by the adenovirus E4-6/7 protein

Schaley J; O'Connor RJ; Taylor LJ; Bar-Sagi D; Hearing P
The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression
PMID: 10666238
ISSN: 0022-538x
CID: 62955

Single cell assays for Rac activity

Taylor LJ; Walsh AB; Hearing P; Bar-Sagi D
PMID: 11036615
ISSN: 0076-6879
CID: 62952