NRF2 Promotes Tumor Maintenance by Modulating mRNA Translation in Pancreatic Cancer

“To determine whether the subset of proteins specifically oxidized in KPn cells represented any particular biochemical processes, the data were analyzed using DAVID (Huang et al., 2009) for pathway enrichment.”

“This is reflected by a short median survival of 6 months and a 5-year survival of 5% (Siegel et al., 2015).”

“This was combined with a parallel analysis of the total proteome by using amine-reactive isobaric tags for relative and absolute quantification (iTRAQ; Ross et al., 2004) (Figure 2A).”

“%) of activating KRASmutations and inactivation of the tumor suppressors TP53, P16/INK4A, and SMAD4 (Jones et al., 2008).”

“…cysteinemodifications, prompting us to further evaluate the functional role of Nrf2 inmRNA translation for pancreatic cancer cells. mRNA Translation Is Impeded in Nrf2-Deficient Cells mRNA translation is a vital cellular process that regulates growth and metabolism (Sonenberg and Hinnebusch, 2009).”

“We previously found that oncogenic Kras expression induced an important regulator of redox control, the transcription factor nuclear factor erythroid-derived 2-like 2, Nfe2l2/Nrf2 (DeNicola et al., 2011).”

“Nrf2 Is a Critical Biological Dependency in Pancreatic Cancer The organoid culture system supports the proliferation of normal andmalignant primary pancreatic ductal cells of bothmouse and human origins (Boj et al., 2015).”

“Hif1a likely contributes to the glycolytic phenotype of PDA (Ying et al., 2012), working in parallel with NRF2 transcriptional target genes that are metabolic enzymes governing the pentose phosphate pathway (Mitsuishi et al., 2012) to promote metabolic rewiring in cancer cells.”

“This includes erastin (Yang and Stockwell, 2008), dehydroascorbic acid (oxidized vitamin C) (Yun et al., 2015), and platinumbased cytotoxics (Socinski, 2004).”

“Reactivation of EGFR and MAPK (mitogen-activated protein kinase) signaling upon inhibition of AKT is an important adaptive survival response, leading to drug resistance in Kras mutant cancer cells, and requires an intact autocrine EGFR signaling cascade to enact (Mendoza et al., 2011).”

“Although oxidative stress may disrupt biological functions, redox reactions in a cell are often tightly regulated and play essential physiological roles (Trachootham et al., 2008).”

“Hif1a likely contributes to the glycolytic phenotype of PDA (Ying et al., 2012), working in parallel with NRF2 transcriptional target genes that are metabolic enzymes governing the pentose phosphate pathway (Mitsuishi et al., 2012) to promote metabolic rewiring in cancer cells.”

“…that the initiation factor eIF3J may also be a candidate redox switch (Figure 3F), we extended our investigation on translation initiation biochemically. eIF4E is the limiting initiation factor for mRNA translation, as it recognizes the m7GTP cap on mRNA and recruits eIF4G (Gingras et al., 1999).”

“Compromised Hif1a protein levels in KPn organoids corresponded with a decrease in the expression level of Hif1a target genes (Figure 5D), their inability to grow in hypoxia (Figure 5E), and heightened sensitivity to inhibition of glycolysis (Figure 5F) (Semenza, 2013).”

“residues oxidized by BSO included a previously reported redoxsensitive protein, Pkm2 (Anastasiou et al., 2011).”

“Metalloproteases of the ADAM (a disintegrin and metalloprotease) family are thought to be responsible for the shedding of certain EGFR ligands, with Adam10 emerging as the main sheddase of EGF (Sahin et al., 2004).”

“…that deregulate mRNA translation are common events in human cancers (Bilanges and Stokoe, 2007), while Cell 166, 963–976, August 11, 2016 ª 2016 Elsevier Inc. 963 enforced expression of certain translation factors transforms rodent fibroblasts and promotes tumorigenesis (Wendel et al., 2004).”

“The concept that elevated levels of free radicals and oxidative stress contributes to DNA damage and predisposition to cancer has led to the recommendation of antioxidant use as prophylaxis against neoplasia (Khansari et al., 2009).”

“This includes erastin (Yang and Stockwell, 2008), dehydroascorbic acid (oxidized vitamin C) (Yun et al., 2015), and platinumbased cytotoxics (Socinski, 2004).”

“, 2007) and various antioxidants have either failed to reduce cancer incidence or have, instead, promoted cancer in clinical and preclinical testing (Sayin et al., 2014).”

“, 2015), as in the case of Krasmutant cells (Bar-Sagi and Feramisco, 1986).”

“This is consistent with earlier reports showing a correlation between the expression of Nrf2 and some of these proteins (Fan et al., 2014; Ke et al., 2013;Malec et al., 2010; Niture and Jaiswal, 2012; Wang et al., 2016).”

“Regarding the potential clinical implications of this work, AKT inhibitors have already shown some preliminary activity in clinical trials (Molife et al., 2014; Yap et al., 2011), and MK2206 is currently being investigated in patients with pancreatic cancer (https://ClinicalTrials.”

“A hallmark of Kras mutant cells is the role of autocrine and paracrine loops in amplifying oncogenic Kras signaling through AKT and ERK (extracellular signal-regulated kinase) (Ardito et al., 2012).”

“Consistently, phosphorylation of eIF4G and eIF4E—substrates of Akt/mTOR (Raught et al., 2000) and Erk/Mnk signaling (Ueda et al., 2004)—respectively, were also decreased in Kn and KPn organoids, aswell as in T organoids expressing shNRF2 (Figures 4C, S4E, andS4F).”

“Alterations in cellular redox levels may cause reversible modifications on specific cysteine residues and alter the activity of their corresponding proteins (Paulsen and Carroll, 2010).”

“We posit this to be due to mTOR-mediated growth suppression in cells that rely on extracellular proteins as an amino acid source (Palm et al., 2015), as in the case of Krasmutant cells (Bar-Sagi and Feramisco, 1986).”

“Sensitivity of Nrf2-deficient cells was selective toward inhibition of PI3K/ AKT, as similar vulnerability was absent toward the MEK (MAPK/ERK kinase) inhibitor AZD6244 (Figures S6P and S6Q) (Davies et al., 2007) or toward a panel of commonly used chemotherapeutics (Figure S6R).”

“Of the four phases of protein synthesis (initiation, elongation, termination, and recycling), initiation is considered to be the rate-limiting step in mRNA translation and is often exploited by cancer cells to support tumorigenesis (Pelletier et al., 2015).”

“Historically, increased cancer cell proliferation has been shown to require increased rates of protein synthesis and ribosome number (Johnson et al., 1975; Zetterberg et al., 1995).”

“Certain cysteine thiols can act as redox switches, since oxidative modifications may impact properties such as catalytic activity or conformation of a protein (Barford, 2004).”

“Similar proliferative defects were also observed in the KRAS and TP53 mutant Suit2 PDA cell line (Moore et al., 2001) (Figure S1C).”

“We found that CPV-IRES-mediated translation was also impaired when NRF2 expression was suppressed (Figure 3C).”

“While HCVIRES-mediated translation requires certain canonical initiation factors (Otto and Puglisi, 2004), the cricket paralysis virus IRES (CPV-IRES) (Wilson et al., 2000) enables translation of mRNAs independently of any translation initiation factors.”

“Consistent with the notion that Nrf2 modulates Akt/mTOR activity downstream of mitogenic signaling, elevated levels of lysosomal hydrolysis were observed in KPn organoids (Figure S6M), and this process was redox dependent (Figures S6N and S6O).”

“This is consistent with earlier reports showing a correlation between the expression of Nrf2 and some of these proteins (Fan et al., 2014; Ke et al., 2013;Malec et al., 2010; Niture and Jaiswal, 2012; Wang et al., 2016).”

“Impaired translation in KPn organoids was not due to eIF2 (eukaryotic initiation factor 2)-mediated activation of the unfolded protein response upon elevated ROS (Figure S3B) (Ling and Söll, 2010).”

“Small molecules that target these pathways suppress mRNA translation and have anti-tumor effects (Choo and Blenis, 2009).”

“However, Nrf2 activators (Talalay et al., 2007) and various antioxidants have either failed to reduce cancer incidence or have, instead, promoted cancer in clinical and preclinical testing (Sayin et al., 2014).”

“PI3K (phosphatidylinositol 3-kinase)/AKT/mTOR signaling has been shown to promote phosphorylation of 4EBPs at multiple sites, resulting in the liberation of eIF4E from4EBP1 and the stimulation of cap-dependent translation initiation (Ruggero and Sonenberg, 2005).”

“Regarding the potential clinical implications of this work, AKT inhibitors have already shown some preliminary activity in clinical trials (Molife et al., 2014; Yap et al., 2011), and MK2206 is currently being investigated in patients with pancreatic cancer (https://ClinicalTrials.gov IDs…”

“To decipher changes in the cysteine proteome, we devised a highly sensitive proteomic method using a selectively cleavable cysteine-reactive (ICAT) reagent to enrich for and identify reduced cysteinecontaining peptides (Sethuraman et al., 2004).”

“For example, Hif1a, an important transcription factor that mediates adaptive responses to intratumoral hypoxia and malignant progression in PDA (Zhao et al., 2014), was markedly decreased in Nrf2-deficient Kras mutant cells.”

“…consistent with earlier reports showing a correlation between the expression of Nrf2 and some of these proteins (Fan et al., 2014; Ke et al., 2013;Malec et al., 2010; Niture and Jaiswal, 2012; Wang et al., 2016). qPCR of polysome fractions revealed an increase in the association of the mRNAs of…”

“This is consistent with earlier reports showing a correlation between the expression of Nrf2 and some of these proteins (Fan et al., 2014; Ke et al., 2013;Malec et al., 2010; Niture and Jaiswal, 2012; Wang et al., 2016).”

“Historically, increased cancer cell proliferation has been shown to require increased rates of protein synthesis and ribosome number (Johnson et al., 1975; Zetterberg et al., 1995).”

“%) oxidized (Brandes et al., 2011).”

“Mutations that deregulate mRNA translation are common events in human cancers (Bilanges and Stokoe, 2007), while”

“However, ectopic expression of the cysteine to aspartic acid oxidation mimic (Permyakov et al., 2012) of the elongation factor eEF2 led to a significant decrease in the rate of nascent protein synthesis when compared to its wild-type counterpart (Figures 3F and S3L).”

“This indicates that Nrf2-dependent decrease in [(35)S]-Met incorporation into proteins was not due to alterations in Met to cysteine exchange through the transsulfuration cycle in response to elevated oxidative stress (Belalcázar et al., 2014).”

“This is consistent with earlier reports showing a correlation between the expression of Nrf2 and some of these proteins (Fan et al., 2014; Ke et al., 2013;Malec et al., 2010; Niture and Jaiswal, 2012; Wang et al., 2016). qPCR of polysome fractions revealed an increase in the association of the…”

“Consistent with reports showing that EGFR signaling is coupled to activation of capdependent translation in EGFR wild-type cells (Patel et al., 2013), EGF supplementation was able to restore defective Akt and Erk activation in KPn organoids (Figure S4N).”