Supplementary Figure S3 from Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers (original) (raw)
Related papers
2023
DMEM or in (BxPC-3, Capan-1, SU.86.86, PK-1) RPMI supplemented with 10% FBS. We have used either DMEM (D6429-Sigma) or RPMI (R8758-Sigma) supplemented with 10% FBS (F4135-Sigma) for culturing the cell lines. HPNE-hTERT cells were cultured in base medium following the instructions from ATCC. In short, the base medium for this cell line is: 75% DMEM without glucose (Sigma Cat #D-5030 with additional 2 mM L-glutamine and 1.5 g/L sodium bicarbonate) and 25% Medium M3 Base (INCELL Corp. Cat #M300F-500). To make the complete growth medium, we added the following components to the base medium: fetal bovine serum 5% (final conc.), 10 g/ml human recombinant EGF (#PHG0311L, Gibco), 5.5 mM D-glucose (1g/L), and 750 g/ml puromycin (Gibco). NRF2 overexpressing stable cell lines were generated by using Capan-1 and BxPC-3 cell lines. Before generating stable cell lines, cells were authenticated via short tandem repeat analysis with resulting allelic profiles matching those found in online databases. Cells were then seeded into six-well plates and incubated overnight at a density that yielded 60-70% confluency at the time of transduction. Media was removed from the cultures and replaced with one milliliter of media containing 8 µg/mL hexadimethrine bromide (Sigma-Aldrich, St. Louis, MO). One
Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy
Molecular Cancer, 2011
Background Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer. Results Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P Conclusions Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.
NRF2 Promotes Tumor Maintenance by Modulating mRNA Translation in Pancreatic Cancer
Cell, 2016
Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.
Oncogenic KRAS confers chemoresistance by upregulating NRF2
Cancer research, 2014
Oncogenic KRAS mutations found in 20% to 30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in a regulatory region in exon 1 of NRF2. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Cotreatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS...
Control of Oxidative Stress in Cancer Chemoresistance: Spotlight on Nrf2 Role
Antioxidants
Chemoresistance represents the main obstacle to cancer treatment with both conventional and targeted therapy. Beyond specific molecular alterations, which can lead to targeted therapy, metabolic remodeling, including the control of redox status, plays an important role in cancer cell survival following therapy. Although cancer cells generally have a high basal reactive oxygen species (ROS) level, which makes them more susceptible than normal cells to a further increase of ROS, chemoresistant cancer cells become highly adapted to intrinsic or drug-induced oxidative stress by upregulating their antioxidant systems. The antioxidant response is principally mediated by the transcription factor Nrf2, which has been considered the master regulator of antioxidant and cytoprotective genes. Nrf2 expression is often increased in several types of chemoresistant cancer cells, and its expression is mediated by diverse mechanisms. In addition to Nrf2, other transcription factors and transcriptiona...
The NRF2/KEAP1 Axis in the Regulation of Tumor Metabolism: Mechanisms and Therapeutic Perspectives
Biomolecules
The NRF2/KEAP1 pathway is a fundamental signaling cascade that controls multiple cytoprotective responses through the induction of a complex transcriptional program that ultimately renders cancer cells resistant to oxidative, metabolic and therapeutic stress. Interestingly, accumulating evidence in recent years has indicated that metabolic reprogramming is closely interrelated with the regulation of redox homeostasis, suggesting that the disruption of NRF2 signaling might represent a valid therapeutic strategy against a variety of solid and hematologic cancers. These aspects will be the focus of the present review.
Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway
Nature, 2013
Cancer cells exhibit metabolic dependencies that distinguish them from their normal counterparts 1. Among these addictions is an increased utilization of the amino acid glutamine (Gln) to fuel anabolic processes 2. Indeed, the spectrum of Gln-dependent tumors and the mechanisms whereby Gln supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of Gln utilization in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumor growth. While most cells utilize glutamate dehydrogenase (GLUD1) to convert Gln-derived glutamate (Glu) into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid (TCA) cycle, PDAC relies on a distinct pathway to fuel the TCA cycle such that Gln-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate (OAA) by aspartate transaminase (GOT1). Subsequently, this OAA is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP + ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly
Oncotarget, 2016
Pancreatic stellate cells (PSC), a prominent stromal cell, contribute to the progression of pancreatic ductal adenocarcinoma (PDAC). We aim to investigate the mechanisms by which PSC promote cell proliferation in PDAC cell lines, BxPC-3 and AsPC-1. PSC-conditioned media (PSC-CM) induced proliferation of these cells in a dose-and time-dependent manner. Nrf2 protein was upregulated and subsequently, its transcriptional activity was increased with greater DNA binding activity and transcription of target genes. Downregulation of Nrf2 led to suppression of PSC-CM activity in BxPC-3, but not in AsPC-1 cells. However, overexpression of Nrf2 alone resulted in increased cell proliferation in both cell lines, and treatment with PSC-CM further enhanced this effect. Activation of Nrf2 pathway resulted in upregulation of metabolic genes involved in pentose phosphate pathway, glutaminolysis and glutathione biosynthesis. Downregulation and inhibition of glucose-6-phosphatedehydrogenase with siRNA and chemical approaches reduced PSC-mediated cell proliferation. Among the cytokines present in PSC-CM, stromal-derived factor-1 alpha (SDF-1α) and interleukin-6 (IL-6) activated Nrf2 pathway to induce cell proliferation in both cells, as shown with neutralization antibodies, recombinant proteins and signaling inhibitors. Taken together, SDF-1α and IL-6 secreted from PSC induced PDAC cell proliferation via Nrf2-activated metabolic reprogramming and ROS detoxification.
Oncogenic KRAS engages an RSK1/NF1 complex in pancreatic cancer
2020
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC.Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways and toxicity due to the targeting of normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil new potential therapeutic targets. Here, we used proximity labelling to identify protein interactors of active KRAS in PDAC cells. Fusions of wildtype (BirA-KRAS4B), mutant (BirA-KRAS4BG12D) and non-transforming and cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase were expressed in murine PDAC cells. Mass spectrometry analysis revealed tha...
Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer
Proceedings of the National Academy of Sciences of the United States of America, 2021
Significance For decades, KRAS interactors have been sought after as potential therapeutic targets in KRAS mutant cancers, especially pancreatic ductal adenocarcinoma (PDAC). Our proximity labeling screen with KRAS in PDAC cells highlights RSK1 as a notable mutant-specific interactor. Functionally, we show that RSK1 mediates negative feedback on wild-type (WT) KRAS in PDAC cells. Targeting oncogenic KRAS eliminates the negative feedback on WT RAS and highlights a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS ablation. Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicit...