Heterogeneity in ERK activity as visualized by in vivo FRET imaging of mammary tumor cells developed in MMTV-Neu mice

Kumagai, Yuka; Naoki, Honda; Nakasyo, Eiji; Kamioka, Yuji; Kiyokawa, Etsuko; Matsuda, Minoru

doi:10.1038/onc.2014.28

Cited by 30 publications

(24 citation statements)

References 36 publications

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“…While ERK activation is widely regarded as pro-tumourigenic, it was recently shown to inversely correlate with stem cell self-renewal in mammary tumours36 and with the maintenance of stem cell identity in mouse intestine37. Also noteworthy in this context, activation of the STAT3 pathway by IL6 (ref.…”

Section: Discussionmentioning

confidence: 99%

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

et al. 2016

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Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, but its molecular heterogeneity hampers the design of targeted therapies. Currently, the only therapeutic option for advanced HCC is Sorafenib, an inhibitor whose targets include RAF. Unexpectedly, RAF1 expression is reduced in human HCC samples. Modelling RAF1 downregulation by RNAi increases the proliferation of human HCC lines in xenografts and in culture; furthermore, RAF1 ablation promotes chemical hepatocarcinogenesis and the proliferation of cultured (pre)malignant mouse hepatocytes. The phenotypes depend on increased YAP1 expression and STAT3 activation, observed in cultured RAF1-deficient cells, in HCC xenografts, and in autochthonous liver tumours. Thus RAF1, although essential for the development of skin and lung tumours, is a negative regulator of hepatocarcinogenesis. This unexpected finding highlights the contribution of the cellular/tissue environment in determining the function of a protein, and underscores the importance of understanding the molecular context of a disease to inform therapy design.

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Section: Discussionmentioning

confidence: 99%

A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis

et al. 2016

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“…Johnsson et al visualized Rac1 signaling during disease development in multiple organs using Rac1-FRET mice (21). Kumagai et al revealed that breast cancer cells with low ERK activity possess cancer stem-cell-like properties by isolating the cells depending on the FRET efficiency from MMTV-Neu:Eisuke mice, a transgenic mouse line expressing the ERK FRET biosensor (36). And in this work, we focus on an example of intravital imaging with the EKAREV-NLS FRET biosensor, which successfully resolved an urgent question about drug resistance in cancer treatment.…”

Section: Application To Intravital Imagingmentioning

confidence: 97%

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

Hirata

Kiyokawa

2016

Biophysical Journal

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Fö rster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.Since the discovery of green fluorescent protein (GFP) (1), scientists have been widely employing this gene-encoded fluorescent protein (FP) to investigate the spatiotemporal dynamics of molecules with subcellular resolution. One of the applications of FP engineering is the development of biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) (2). FRET is a nonradiative energy transfer process between two fluorophores located in close proximity to each other, and its efficiency is strongly dependent on the distance between the fluorophores. More precisely, the energy transfer rate (k t ) from a donor to an acceptor fluorophore is calculated in the following Förster's equation;where J is the overlap of donor emission and acceptor excitation spectra, k 2 is an orientation factor of transition moment (a factor determined by the relative orientation of the two fluorophores), n is a refractive index, R is the distance between the donor and acceptor fluorophores, and k f is the donor fluorescence emission speed. Therefore, FRET efficiency is determined by the distance and orientation of the two fluorophores. To measure FRET efficiency, there are roughly two methods; ratiometric analysis and lifetime analysis. Ratiometric analysis utilizes fluorescence intensity of an acceptor (e.g., yellow FP (YFP)) and a donor (e.g., cyan FP (CFP)) upon the donor excitation. Because accept...

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“…Similarly, the Erk and PAK-FRET biosensor mice have permitted us to untangle the molecular mechanisms governing extracellular signal transduction, proliferation, and differentiation in vivo (14)(15)(16). Interestingly, the Erk FRET mouse has recently been used to study Erk activity in distinct subpopulations of breast cancer cells to monitor chemotherapy pharmacokinetics in breast cancer (17). FRET biosensors are, therefore, an emerging preclinical tool for cancer research and help us probe dynamic events in vivo rather than the current in vitro static and fixed endpoints discussed earlier.…”

Section: Discussionmentioning

confidence: 99%