Src Family Kinase Inhibitors Block Amphiregulin-Mediated Autocrine ErbB Signaling in Normal Human Keratinocytes

Kansra, Sanjay; Stoll, Stefan; Johnson, Jessica L.; Elder, James T.

doi:10.1124/mol.104.004689

Cited by 27 publications

(20 citation statements)

References 59 publications

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“…Dsg2 did not affect total c-Src; however, activated P-c-Src (Tyr416) was dramatically reduced in the Dsg2 KD cells (Figure 5A). Inhibition of c-Src with the inhibitor PP2 partially abrogated phosphorylation of EGFR in response to EGF ligand in HaCaT cells (Figure 5B), confirming previous findings that c-Src acts both upstream as well as downstream of EGFR [47]. Thus, the Dsg2-dependent EGFR activation may be modulated, in part, by c-Src.…”

Section: Resultssupporting

confidence: 88%

c-Src/Cav1-dependent activation of the EGFR by Dsg2

et al. 2016

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The desmosomal cadherin, desmoglein 2 (Dsg2), is deregulated in a variety of human cancers including those of the skin. When ectopically expressed in the epidermis of transgenic mice, Dsg2 activates multiple mitogenic signaling pathways and increases susceptibility to tumorigenesis. However, the molecular mechanism responsible for Dsg2-mediated cellular signaling is poorly understood. Here we show overexpression as well as co-localization of Dsg2 and EGFR in cutaneous SCCs in vivo. Using HaCaT keratinocytes, knockdown of Dsg2 decreases EGFR expression and abrogates the activation of EGFR, c-Src and Stat3, but not Erk1/2 or Akt, in response to EGF ligand stimulation. To determine whether Dsg2 mediates signaling through lipid microdomains, sucrose density fractionation illustrated that Dsg2 is recruited to and displaces Cav1, EGFR and c-Src from light density lipid raft fractions. STED imaging confirmed that the presence of Dsg2 disperses Cav1 from the cell-cell borders. Perturbation of lipid rafts with the cholesterol-chelating agent MβCD also shifts Cav1, c-Src and EGFR out of the rafts and activates signaling pathways. Functionally, overexpression of Dsg2 in human SCC A431 cells enhances EGFR activation and increases cell proliferation and migration through a c-Src and EGFR dependent manner. In summary, our data suggest that Dsg2 stimulates cell growth and migration by positively regulating EGFR level and signaling through a c-Src and Cav1-dependent mechanism using lipid rafts as signal modulatory platforms.

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Section: Resultssupporting

confidence: 88%

c-Src/Cav1-dependent activation of the EGFR by Dsg2

et al. 2016

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“…Shed AREG in keratinocyte-conditioned medium or cell-associated AREG in radio immunoprecipitation assay buffer (RIPA) lysates were measured by sandwich ELISA (R&D Systems) using anti-AREG antibodies (Kansra et al , 2005; Stoll et al , 2010b). …”

Section: Methodsmentioning

confidence: 99%

Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation

Stoll

Stuart

Lambert

et al. 2016

Journal of Investigative Dermatology

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The EGF receptor (EGFR) and its ligands are essential regulators of epithelial biology, which are often amplified in cancer cells. We have previously shown that shRNA-mediated silencing of one of these ligands, amphiregulin (AREG), results in keratinocyte growth arrest that cannot be rescued by soluble extracellular EGFR ligands. To further explore the functional importance of specific AREG domains, we stably transduced keratinocytes expressing tetracycline-inducible AREG-targeted shRNA with lentiviruses expressing silencing-proof, membrane-tethered AREG cytoplasmic and extracellular domains (AREG-CTD and AREG-ECD), as well as full-length AREG precursor (proAREG). Here we show that growth arrest of AREG-silenced keratinocytes occurs in G2/M and is significantly restored by proAREG and AREG-CTD, but not by AREG-ECD. Moreover, the AREG-CTD was sufficient to normalize cell cycle distribution profiles and expression of mitosis-related genes. Our findings uncover an important role of the AREG-CTD in regulating cell division, which may be relevant to tumor resistance to EGFR-directed therapies.

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“…2 Epidermal growth factor receptor (EGFR) signaling is an important regulator of epidermal development and homeostasis 3–6 and plays an important role in the control of multiple keratinocyte functions including migration, 7,8 proliferation, 9 differentiation, 10–12 survival 13,14 and activation of innate and adaptive immune responses. 15–17 Deregulated EGFR signaling is a hallmark of epithelial cancer 18 , and EGFR is a validated target in several types of carcinomas.…”

Section: Introductionmentioning

confidence: 99%

The EGF receptor ligand amphiregulin controls cell division via FoxM1

et al. 2015

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Epidermal growth factor receptor (EGFR) is central to epithelial cell physiology and deregulated EGFR signaling has a key role in a variety of human carcinomas. Here we show that silencing of the EGF-related factor amphiregulin (AREG) markedly inhibits the expansion of human keratinocytes through mitotic failure and accumulation of cells with ≥4n DNA content. RNA-seq-based transcriptome analysis revealed that tetracycline-mediated AREG silencing significantly altered the expression of 2,331 genes, 623 of which were not normalized by treatment with EGF. Interestingly, genes irreversibly up-regulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. Moreover, a significant proportion of the irreversibly down-regulated genes featured upstream binding sites recognized by FoxM1, a key transcription factor in the control of mitosis that is widely dysregulated in cancer. The downregulation of FoxM1 and its target genes preceded mitotic arrest. Constitutive expression of FoxM1 in AREG knockdown cells normalized cell proliferation, reduced the number of cells with ≥4n DNA content, and rescued expression of FoxM1 target genes. These results demonstrate that AREG controls G2/M progression and cytokinesis in keratinocytes via activation of a FoxM1-dependent transcriptional program, suggesting new avenues for treatment of epithelial cancer.

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Src Family Kinase Inhibitors Block Amphiregulin-Mediated Autocrine ErbB Signaling in Normal Human Keratinocytes

Cited by 27 publications

References 59 publications

c-Src/Cav1-dependent activation of the EGFR by Dsg2

c-Src/Cav1-dependent activation of the EGFR by Dsg2

Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation

The EGF receptor ligand amphiregulin controls cell division via FoxM1

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