Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

Augsten, Martin; Böttcher, Anika; Spanbroek, Rainer; Rubio, Ignacio; Friedrich, Karlheinz

doi:10.1186/1478-811x-12-1

Cited by 40 publications

(40 citation statements)

References 62 publications

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“…To quantify cell invasion, we measure wound confluence using a modified scratch wound invasion assay where cells are overlaid with a ~1.5 mm-thick layer of Matrigel, a protein mixture that recapitulates the ECM (Fig 1B); this setup requires cells to invade through a 3D matrix 30,31 . Our results show that there is variability in how quickly pancreatic ductal cells move into the wound gap.…”

Section: Resultsmentioning

confidence: 99%

“…To measure cell invasion through a 3D matrix, we perform modified scratch wound invasion assays with an overlay of Matrigel to simulate the ECM 30,31,84 . We plate cells in the wells of a 96-well plate with a thin Matrigel (100 μg/ml) layer for cell attachment at 95% confluency, create a scratch wound, overlay the scratch with a ~1.5 mm-thick layer of 8 mg/ml Matrigel (Corning), and perform time-lapse imaging using the IncuCyte Zoom (Essen Bioscience) at 5% CO 2 and 37°C.…”

Section: Experimental Methodsmentioning

confidence: 99%

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Stiffness of pancreatic cancer cells is associated with increased invasive potential

et al. 2016

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Metastasis is a fundamentally physical process in which cells are required to deform through narrow gaps as they invade surrounding tissues and transit to distant sites. In many cancers, more invasive cells are more deformable than less invasive cells, but the extent to which mechanical phenotype, or mechanotype, can predict disease aggressiveness in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here we investigate the invasive potential and mechanical properties of immortalized PDAC cell lines derived from primary tumors and a secondary metastatic site, as well as noncancerous pancreatic ductal cells. To investigate how invasive behavior is associated with cell mechanotype, we flow cells through micron-scale pores using parallel microfiltration and microfluidic deformability cytometry; these results show that the ability of PDAC cells to passively transit through pores is only weakly correlated with their invasive potential. We also measure the Young’s modulus of pancreatic ductal cells using atomic force microscopy, which reveals that there is a strong association between cell stiffness and invasive potential in PDAC cells. To determine the molecular origins of the variability in mechanotype across our PDAC cell lines, we analyze RNAseq data for genes that are known to regulate cell mechanotype. Our results show that vimentin, actin, and lamin A are among the most differentially expressed mechanoregulating genes across our panel of PDAC cell lines, as well as a cohort of 38 additional PDAC cell lines. We confirm levels of these proteins across our cell panel using immunoblotting, and find that levels of lamin A increase with both invasive potential and Young’s modulus. Taken together, we find that stiffer PDAC cells are more invasive than more compliant cells, which challenges the paradigm that decreased cell stiffness is a hallmark of metastatic potential.

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Section: Resultsmentioning

confidence: 99%

Section: Experimental Methodsmentioning

confidence: 99%

Stiffness of pancreatic cancer cells is associated with increased invasive potential

et al. 2016

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“…While a subset of these miRNAs are overexpressed in cancers and shown to target tumor suppressor genes, hence termed oncogenic miRNAs or oncomiRs, some other miRNAs that target oncogenes are underexpressed in cancers, which are often called tumor-suppressor-like miRNAs. Certain miRNAs are known to have a dual role, either oncogenic or tumor-suppressive, depending on the cancer-specific context [22] .…”

Section: Introductionmentioning

confidence: 99%

Emerging Role of MicroRNAs in mTOR Signaling

Zhang

Huang

Wang³

et al. 2017

Cell. Mol. Life Sci.

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Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase that plays a critical role in the control of cellular growth and metabolism. Hyperactivation of mTOR pathway is common in human cancers, driving uncontrolled proliferation. MicroRNA (miRNA) is a class of short noncoding RNAs that regulate the expression of a wide variety of genes. Deregulation of miRNAs is a hallmark of cancer. Recent studies have revealed interplays between miRNAs and the mTOR pathway during cancer development. Such interactions appear to provide a fine-tuning of various cellular functions and contribute qualitatively to the behavior of cancer. Here we provide an overview of current knowledge regarding the reciprocal relationship between miRNAs and mTOR pathway: regulation of mTOR signaling by miRNAs and control of miRNA biogenesis by mTOR. Further research in this area may prove important for the diagnosis and therapy of human cancer.

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“…A plethora of studies has categorically shown that oncogenic Ras triggers cell cycle entry and DNA synthesis independently of growth factors [1,2]. Accordingly, inhibition of native Ras by a variety of approaches including microinjection of inhibitory Abs [3], dominant negative RasS17N [4], expression of Ras-GTP scavenging modules [5], small-molecular allosteric inhibitors of Ras function [6] or by preventing upstream steps in Ras activation [7] inhibits DNA synthesis and cell proliferation. More recently, the analysis of engineered MEF lines devoid of all 3 Ras genes (termed Rasless) has underscored the indispensable nature of Ras in cell cycle progression, as cells rendered Rasless were forced into a quiescencelike state and ceased to proliferate [8,9].…”

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confidence: 99%

Ras signals principally via Erk in G1 but cooperates with PI3K/Akt for Cyclin D induction and S-phase entry

et al. 2019

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Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.

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Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

Cited by 40 publications

References 62 publications

Stiffness of pancreatic cancer cells is associated with increased invasive potential

Stiffness of pancreatic cancer cells is associated with increased invasive potential

Emerging Role of MicroRNAs in mTOR Signaling

Ras signals principally via Erk in G1 but cooperates with PI3K/Akt for Cyclin D induction and S-phase entry

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