Oncogenic mutant RAS signaling activity is rescaled by the ERK/MAPK pathway

Gillies, Taryn E.; Pargett, Michael; Silva, Jillian M.; Teragawa, Carolyn K.; McCormick, Frank; Albeck, John G.

doi:10.1101/2020.02.17.952093

Cited by 6 publications

(8 citation statements)

References 46 publications

(60 reference statements)

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“…We titrated the virus to allow ectopic expression of KRAS G12D within 2-to 4-fold of endogenous levels (Figures S5B and S5C) because a higher level of KRAS overexpression was not tolerated by these organoids (Figure S5D). In acinus-like and duct-like organoids, KRAS G12D expression induced a modest increase in phosphorylation of mitogen-activated protein kinase (p-ERK) (Figures S5B and S5C), consistent with previous studies (Gillies et al, 2020;Tuveson et al, 2004). Expression of KRAS G12D was induced on day 8 after cells were committed to a ductlike or acinus-like lineage, and the phenotypes were analyzed 16 days later.…”

Section: Temporal Changes In Marker Expression During Ductlike and Acinus-like Lineage Specificationsupporting

confidence: 85%

Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

Huang¹,

Desai²,

Conrad³

et al. 2021

Cell Stem Cell

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Summary The exocrine pancreas, consisting of ducts and acini, is the site of origin of pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Our understanding of the genesis and progression of human pancreatic diseases, including PDAC, is limited because of challenges in maintaining human acinar and ductal cells in culture. Here we report induction of human pluripotent stem cells toward pancreatic ductal and acinar organoids that recapitulate properties of the neonatal exocrine pancreas. Expression of the PDAC-associated oncogene GNAS R201C induces cystic growth more effectively in ductal than acinar organoids, whereas KRAS G12D is more effective in modeling cancer in vivo when expressed in acinar compared with ductal organoids. KRAS G12D , but not GNAS R201C , induces acinar-to-ductal metaplasia-like changes in culture and in vivo . We develop a renewable source of ductal and acinar organoids for modeling exocrine development and diseases and demonstrate lineage tropism and plasticity for oncogene action in the human pancreas.

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Section: Temporal Changes In Marker Expression During Ductlike and Acinus-like Lineage Specificationsupporting

confidence: 85%

Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

Huang¹,

Desai²,

Conrad³

et al. 2021

Cell Stem Cell

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“…For the AMPKAR2 reporter, AMPKAR2 phosphorylation status was calculated using the protocol described in (Kosaisawe et al, 2021). Briefly, linearized AMPKAR2 FRET efficiency was calculated as shown in our previous work (Gillies et al, 2020). Then AMPKAR2 phosphorylation status was estimated based on this equation…”

Section: Reporter Activity Analysismentioning

confidence: 99%

Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis

Sparta

Pargett

Kosaisawe

et al. 2021

Preprint

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mTORC1 senses nutrient and growth factor status and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. The molecular mechanisms of mTORC1 activation are thought to enforce a strict requirement for simultaneous amino acid and growth factor stimuli, but this model has not been evaluated with quantitative or single-cell methods. Here, we develop a series of fluorescent protein-TFEB fusions and investigate how combinations of stimuli jointly regulate signaling from mTORC1 to TFEB at the single-cell level. Live-cell imaging of individual cells revealed that mTORC1-TFEB signaling responds with graded changes to individual amino acid and growth factor inputs, rather than behaving as a logical AND gate. We find that mTORC1 inputs can be sequentially sensed, with responses that vary between mTORC1 substrates and are amplified by input from other kinases, including GSK3β. In physiologically relevant concentrations of amino acids, we observe fluctuations in mTORC1-TFEB signaling that indicate continuous responsiveness to nutrient availability. Our results clarify how the molecular regulation of mTORC1 enables homeostatic processes at the cellular level and provide a more precise understanding of its behavior as an integrator of multiple inputs.

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“…It is made The copyright holder for this preprint this version posted March 4, 2021. ; https://doi.org/10.1101/2021.03.04.433941 doi: bioRxiv preprint negative DUSP6 mutant C293S, the surviving subpopulations appear to harbor even more p-ERK (40). Thus, cells with RAS mutations adjust their feedback signaling to constrain p-ERK, and long-term selective pressure can lead to genetic or epigenetic changes that allow cells to benefit from reduced DUSP6 activity and increased p-ERK (31,38,40). In our LUAD cells with reintroduction of NKX2-1, we found that the p-ERK induced by stimulation with RAS pathway agonists EGF or PMA trended lower in cells expressing NKX2-1 compared to cells with empty vector (Fig.…”

Section: Nkx2-1 Inhibits Cell Proliferation Migration and Invasionmentioning

confidence: 99%

“…ERK mediates negative feedback signaling to multiple upstream components of the RAS pathway. In this way, cellular ERK activity is highly constrained, even in cells expressing mutant, constitutively active KRAS (31). In vivo, release from negative feedback loops allows for elevated ERK signaling that drives malignant progression (32)(33)(34).…”

Section: Introductionmentioning

confidence: 99%

NKX2-1 controls lung cancer progression by inducing DUSP6 to dampen ERK activity

Ingram

Samson

Zewdu

et al. 2021

Preprint

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Lung cancer remains a leading cause of cancer death, with unclear mechanisms driving the transition to aggressive cancer with poor prognosis. The RAS→RAF→MEK→ERK pathway is hyper-activated in ~50% of human lung adenocarcinoma (LUAD). An initial activating mutation induces homeostatic feedback mechanisms that limit ERK activity. Additional, undefined genetic hits overcome the feedback, leading to high ERK activity that drives malignant progression. At detection, the majority of LUADs express the homeobox transcription factor NKX2-1, which also limits malignant progression. NKX2-1 constrains LUAD, in part, by maintaining a well-differentiated state with features of pulmonary identity. We asked if loss of NKX2-1 might also contribute to the release of ERK activity that drives tumor progression. Using human tissue samples and cell lines, xenografts, and genetic mouse models, we show that NKX2-1 induces the ERK phosphatase DUSP6. In tumor cells from late-stage LUAD with silenced NKX2-1, re-introduction of NKX2-1 induces DUSP6 and inhibits cell proliferation and migration and tumor growth and metastasis. CRISPR knockout studies show that DUSP6 is necessary for NKX2-1-mediated inhibition of tumor progression in vivo. Further, DUSP6 expression is sufficient to inhibit RAS-driven LUAD. We conclude that NKX2-1 silencing, and thereby DUSP6 downregulation, is a mechanism by which early LUAD can unleash ERK hyperactivation for tumor progression.

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Oncogenic mutant RAS signaling activity is rescaled by the ERK/MAPK pathway

Cited by 6 publications

References 46 publications

Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis

NKX2-1 controls lung cancer progression by inducing DUSP6 to dampen ERK activity

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