scholarly journals Oncogenic KRAS engages an RSK1/NF1 complex in pancreatic cancer

2020 ◽  
Author(s):  
Derek K. Cheng ◽  
Tobiloba E. Oni ◽  
Youngkyu Park ◽  
Jennifer S. Thalappillil ◽  
Hsiu-Chi Ting ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Clinical Success ◽  
Treatment Options ◽  
Therapeutic Targets ◽  
Mass Spectrometry Analysis ◽  
Ductal Adenocarcinoma ◽  
Wild Type ◽  
Additional Function ◽  
Feedback Mechanisms ◽  
Spectrometry Analysis

AbstractPancreatic 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 that RSK1 was enriched among proteins that selectively interacted with membrane-bound KRASG12D. RSK1 required the NF1 and SPRED proteins to interact with KRAS-GTP at the membrane. In both murine and human PDAC lines, membrane-targeted RSK1 was tolerated but inhibited cell proliferation following oncogenic KRAS abrogation to reveal a negative feedback role for membrane-localized RSK1 on wild-type KRAS. Inhibition of the wild-type KRAS, which has been previously proposed to suppress KRAS oncogenesis, may partially explain how RSK1 has been identified as a dependency in some KRAS mutant cells and may provide an additional function for NF1 in tumorigenesis.Significance StatementFor decades, KRAS interactors have been sought after as potential therapeutic targets in KRAS mutant cancers, especially pancreatic adenocarcinoma (PDAC). Our proximity labeling screen with KRAS in PDAC cells highlight RSK1 as a notable mutant-specific interactor. Functionally, we show that RSK1 mediates negative feedback on wild-type KRAS in PDAC cells.

scholarly journals Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer

2021 ◽  
Vol 118 (21) ◽  
pp. e2016904118
Author(s):  
Derek K. Cheng ◽  
Tobiloba E. Oni ◽  
Jennifer S. Thalappillil ◽  
Youngkyu Park ◽  
Hsiu-Chi Ting ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Clinical Success ◽  
Treatment Options ◽  
Mass Spectrometry Analysis ◽  
Ductal Adenocarcinoma ◽  
Ras Signaling ◽  
Wild Type ◽  
Feedback Mechanisms ◽  
Spectrometry Analysis ◽  
Pancreatic Cancer Cells

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 toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

scholarly journals Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

2010 ◽  
Vol 192 (8) ◽  
pp. 2044-2052 ◽  
Author(s):  
Jyl S. Matson ◽  
Hyun Ju Yoo ◽  
Kristina Hakansson ◽  
Victor J. DiRita
Keyword(s):  
Antimicrobial Peptides ◽  
Vibrio Cholerae ◽  
Lipid A ◽  
Polymyxin B ◽  
Mass Spectrometry Analysis ◽  
Peptide Antibiotic ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Antimicrobial Peptide Resistance ◽  
El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

scholarly journals Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

2010 ◽  
Vol 192 (18) ◽  
pp. 4651-4659 ◽  
Author(s):  
Wendy D. Smith ◽  
Jonathan A. Pointon ◽  
Emily Abbot ◽  
Hae Joo Kang ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Human Keratinocytes ◽  
Mass Spectrometry Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Protein Adhesion ◽  
Culture Supernatants

ABSTRACT Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

scholarly journals Modification of Lipooligosaccharide with Phosphoethanolamine by LptA in Neisseria meningitidis Enhances Meningococcal Adhesion to Human Endothelial and Epithelial Cells

2008 ◽  
Vol 76 (12) ◽  
pp. 5777-5789 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Russel W. Carlson ◽  
Artur Muszynski ◽  
Biswa Choudhury ◽  
Kwang Sik Kim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neisseria Meningitidis ◽  
Lipid A ◽  
Inner Core ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Epithelial Cell Lines

ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.

scholarly journals Single Nucleotide Polymorphisms in MIR143 Contribute to Protection Against Non-Hodgkin Lymphoma (NHL) in Caucasian Populations

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 185 ◽  
Author(s):  
Gabrielle Bradshaw ◽  
Larisa M. Haupt ◽  
Eunise M. Aquino ◽  
Rodney A. Lea ◽  
Heidi G. Sutherland ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Multiple Testing ◽  
Genome Wide Association Study ◽  
Treatment Options ◽  
Significant Snps ◽  
Mass Spectrometry Analysis ◽  
Nucleotide Polymorphisms ◽  
Spectrometry Analysis ◽  
Non Hodgkin Lymphoma ◽  
Reduced Risk

Recent studies show an association of microRNA (miRNA) polymorphisms (miRSNPs) in different cancer types, including non-Hodgkin lymphoma (NHL). The identification of miRSNPs that are associated with NHL susceptibility may provide biomarkers for early diagnosis and prognosis for patients who may not respond well to current treatment options, including the immunochemotherapy drug combination that includes rituximab, cyclophosphamide, doxorubicin, vincristine and prednisome (R-CHOP). We developed a panel of miRSNPs for genotyping while using multiplex PCR and chip-based mass spectrometry analysis in an Australian NHL case-control population (300 cases, 140 controls). Statistical association with NHL susceptibility was performed while using Chi-square (χ2) and logistic regression analysis. We identified three SNPs in MIR143 that are to be significantly associated with reduced risk of NHL: rs3733846 (odds ratio (OR) [95% confidence interval (CI)] = 0.54 [0.33 – 0.86], p = 0.010), rs41291957 (OR [95% CI] = 0.61 [0.39 – 0.94], p = 0.024), and rs17723799 (OR [95% CI] = 0.43 [0.26 – 0.71], p = 0.0009). One SNP, rs17723799, remained significant after correction for multiple testing (p = 0.015). Subsequently, we investigated an association between the rs17723799 genotype and phenotype by measuring target gene Hexokinase 2 (HKII) expression in cancer cell lines and controls. Our study is the first to report a correlation between miRSNPs in MIR143 and a reduced risk of NHL in Caucasians, and it is supported by significant SNPs in high linkage disequilibrium (LD) in a large European NHL genome wide association study (GWAS) meta-analysis.

Impact and mechanistic role of MicroRNA-1291 on pancreatic tumorigenesis.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 243-243 ◽  
Author(s):  
Mei-Juan Tu ◽  
Yu-Zhuo Pan ◽  
Jing-Xin Qiu ◽  
Edward Jae-hoon Kim ◽  
Aiming Yu
Keyword(s):  
Cell Cycle ◽  
Cancer Biology ◽  
Critical Role ◽  
Experimental Studies ◽  
Mass Spectrometry Analysis ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Spectrometry Analysis

243 Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death. Better understanding of pancreatic cancer biology and identification of new targets are highly warranted. MicroRNAs (miRs or miRNAs) play a critical role in the control of tumor progression via crosstalk with cancer signaling pathways. Our recent studies showed that miR-1291 improved chemosensitivity through targeting of efflux transporter ABCC1. This current study investigated the mechanistic role of miR-1291 in the suppression of pancreatic tumorigenesis. Methods: PANC-1 and AsPC-1 cell lines were stably transfected with miR-1291. Cell cycle status and apoptosis of stable miR-1291-expressing cells were tested against control cells using flow cytometry. Cells were injected subcutaneously into nude mice and tumorigenesis was measured in vivo. Proteomic studies were performed by two-dimensional difference gel electrophoresis, matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis. Computationally predicted miR-1291 targets were assessed by luciferase reporter assay and Western blot. Primary PDAC and control samples were tested for miR-1291 and target gene expression levels. Results: Our data showed that stable miR-1291-expressing PANC-1 and AsPC-1 cells both showed a significantly lower rate of proliferation than the control cells, which was associated with a cell cycle arrest and enhanced apoptosis. Furthermore, miR-1291 suppressed the tumorigenesis of PANC-1 cells in mouse models in vivo. Proteomic studies revealed the protein level of several cancer-related genes were downregulated by miR-1291, including a pancreatic tumor promoting protein AGR2 which was reduced ~10-fold. Through computational and experimental studies we further identified that FOXA2, a transcription factor governing AGR2 expression, was a direct target of miR-1291. In addition, we found a significant down-regulation of miR-1291 in a set of PDAC patient tumor samples overexpressing AGR2. Conclusions: These results indicate that miR-1291 suppresses pancreatic tumorigenesis via targeting of FOXA2-AGR regulatory pathway providing new insight supporting development of miR-1291-based therapy for PDAC.

scholarly journals Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Valentina M. T. Mayer ◽  
Markus B. Tomek ◽  
Rudolf Figl ◽  
Marina Borisova ◽  
Isabel Hottmann ◽  
...  
Keyword(s):  
Natural Habitat ◽  
Indirect Evidence ◽  
Mass Spectrometry Analysis ◽  
Membrane Transporter ◽  
Wild Type ◽  
Tannerella Forsythia ◽  
Inner Membrane ◽  
Oral Pathogen ◽  
Spectrometry Analysis ◽  
E Coli

Abstract Background The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T. forsythia, an AmpG-like permease (Tanf_08365) is involved in cytoplasmic muropeptide uptake. In E. coli, AmpG is specific for the import of N-acetylglucosamine (GlcNAc)-anhydroMurNAc(−peptides) which are common PGN turnover products, with the disaccharide portion as a minimal requirement. Currently, it is unclear which natural, complex MurNAc sources T. forsythia can utilize and which role AmpG plays therein. Results We performed a screen of various putative MurNAc sources for T. forsythia mimicking the situation in the natural habitat and compared bacterial growth and cell morphology of the wild-type and a mutant lacking AmpG (T. forsythia ΔampG). We showed that supernatants of the oral biofilm bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and of E. coli ΔampG, as well as isolated PGN and defined PGN fragments obtained after enzymatic digestion, namely GlcNAc-anhydroMurNAc(−peptides) and GlcNAc-MurNAc(−peptides), could sustain growth of T. forsythia wild-type, while T. forsythia ΔampG suffered from growth inhibition. In supernatants of T. forsythia ΔampG, the presence of GlcNAc-anhMurNAc and, unexpectedly, also GlcNAc-MurNAc was revealed by tandem mass spectrometry analysis, indicating that both disaccharides are substrates of AmpG. The importance of AmpG in the utilization of PGN fragments as MurNAc source was substantiated by a significant ampG upregulation in T. forsythia cells cultivated with PGN, as determined by quantitative real-time PCR. Further, our results indicate that PGN-degrading amidase, lytic transglycosylase and muramidase activities in a T. forsythia cell extract are involved in PGN scavenging. Conclusion T. forsythia metabolizes intact PGN as well as muropeptides released from various bacteria and the bacterium’s inner membrane transporter AmpG is essential for growth on these MurNAc sources, and, contrary to the situation in E. coli, imports both, GlcNAc-anhMurNAc and GlcNAc-MurNAc fragments.

scholarly journals Loss of p53 tumor suppression function drives invasion and genomic instability in models of murine pancreatic cancer

2022 ◽  
Author(s):  
Claudia Tonelli ◽  
Astrid Deschênes ◽  
Melissa A. Yao ◽  
Youngkyu Park ◽  
David A. Tuveson
Keyword(s):  
Pancreatic Cancer ◽  
Mouse Model ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Treatment Options ◽  
Intraepithelial Neoplasia ◽  
Ductal Adenocarcinoma ◽  
Wild Type

Pancreatic ductal adenocarcinoma (PDA) is a deadly disease with few treatment options. There is an urgent need to better understand the molecular mechanisms that drive disease progression, with the ultimate aim of identifying early detection markers and clinically actionable targets. To investigate the transcriptional and morphological changes associated with pancreatic cancer progression, we analyzed the KrasLSLG12D/+; Trp53LSLR172H/+; Pdx1-Cre (KPC) mouse model. We have identified an intermediate cellular event during pancreatic carcinogenesis in the KPC mouse model of PDA that is represented by a subpopulation of tumor cells that express KrasG12D, p53R172H and one allele of wild-type Trp53. In vivo, these cells represent a histological spectrum of pancreatic intraepithelial neoplasia (PanIN) and acinar-to-ductal metaplasia (ADM) and rarely proliferate. Following loss of wild-type p53, these precursor lesions undergo malignant de-differentiation and acquire invasive features. We have established matched organoid cultures of pre-invasive and invasive cells from murine PDA. Expression profiling of the organoids led to the identification of markers of the pre-invasive cancer cells in vivo and mechanisms of disease aggressiveness.

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