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Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

ABSTRACT: Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein-tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from casitas B-lineage lymphoma-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy.

SUBMITTER: Li X

PROVIDER: S-EPMC3326223 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

Li X X Lu Y Y Liang K K Hsu J-M JM Albarracin C C Mills G B GB Hung M-C MC Fan Z Z

Oncogene 20120109 40

Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein-tyrosine kinase highly expressed in most human breast tumors, interact ...[more]

PMID: 22231447

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Project description:Resistance to anoikis, cell death due to matrix detachment, is acquired during tumor progression. The 14-3-3σ protein is implicated in the development of chemo- and radiation resistance, indicating a poor prognosis in multiple human cancers. However, its function in anoikis resistance and metastasis in hepatocellular carcinoma (HCC) is currently unknown. Methods: Protein expression levels of 14-3-3σ were measured in paired HCC and normal tissue samples using western blot and immunohistochemical (IHC) staining. Statistical analysis was performed to evaluate the clinical correlation between 14-3-3σ expression, clinicopathological features, and overall survival. Artificial modulation of 14-3-3σ (downregulation and overexpression) was performed to explore the role of 14-3-3σ in HCC anoikis resistance and tumor metastasis in vitro and in vivo. Association of 14-3-3σ with epidermal growth factor receptor (EGFR) was assayed by co-immunoprecipitation. Effects of ectopic 14-3-3σ expression or knockdown on EGFR signaling, ligand-induced EGFR degradation and ubiquitination were examined using immunoblotting and co-immunoprecipitation, immunofluorescence staining, and flow cytometry analysis. The levels of EGFR ubiquitination, the interaction between EGFR and 14-3-3σ, and the association of EGFR with c-Cbl after EGF stimulation, in 14-3-3σ overexpressing or knockdown cells were examined to elucidate the mechanism by which 14-3-3σ inhibits EGFR degradation. Using gain-of-function or loss-of-function strategies, we further investigated the role of the EGFR signaling pathway and its downstream target machinery in 14-3-3σ-mediated anoikis resistance of HCC cells. Results: We demonstrated that 14-3-3σ was upregulated in HCC tissues, whereby its overexpression was correlated with aggressive clinicopathological features and a poor prognosis. In vitro and in vivo experiments indicated that 14-3-3σ promoted anoikis resistance and metastasis of HCC cells. Mechanistically, we show that 14-3-3σ can interact with EGFR and significantly inhibit EGF-induced degradation of EGFR, stabilizing the activated receptor, and therefore prolong the activation of EGFR signaling. We demonstrated that 14-3-3σ downregulated ligand-induced EGFR degradation by inhibiting EGFR-c-Cbl association and subsequent c-Cbl-mediated EGFR ubiquitination. We further verified that activation of the ERK1/2 pathway was responsible for 14-3-3σ-mediated anoikis resistance of HCC cells. Moreover, EGFR inactivation could reverse the 14-3-3σ-mediated effects on ERK1/2 phosphorylation and anoikis resistance. Expression of 14-3-3σ and EGFR were found to be positively correlated in human HCC tissues. Conclusions: Our results indicate that 14-3-3σ plays a pivotal role in the anoikis resistance and metastasis of HCC cells, presumably by inhibiting EGFR degradation and regulating the activation of the EGFR-dependent ERK1/2 pathway. To our best knowledge, this is the first report of the role of 14-3-3σ in the anoikis resistance of HCC cells, offering new research directions for the treatment of metastatic cancer by targeting 14-3-3σ.

Project description:In gut epithelium, IL-22 transmits signals through STAT3 phosphorylation (pSTAT3) which provides intestinal immunity. Many components in the IL-22-pSTAT3 pathway have been identified as risk factors for inflammatory bowel disease (IBD) and some of them are considered as promising therapeutic targets. However, new perspectives are still needed to understand IL-22-pSTAT3 signaling for effective clinical interventions in IBD patients. Here, we revealed activating transcription factor 3 (ATF3), recently identified to be upregulated in patients with active IBD, as a crucial player in the epithelial IL-22-pSTAT3 signaling cascade. We found ATF3 is central to intestinal homeostasis and provides protection during colitis. Loss of ATF3 led to decreased crypt numbers, more shortened colon length, impaired ileal fucosylation at the steady state, and lethal disease activity during DSS-induced colitis which can be effectively ameliorated by rectal transplantation of wild-type colonic organoids. Epithelial stem cells and Paneth cells form a niche to orchestrate epithelial regeneration and host-microbe interactions, and IL-22-pSTAT3 signaling is a key guardian for this niche. We found ATF3 is critical for niche maintenance as ATF3 deficiency caused compromised stem cell growth and regeneration, as well as Paneth cell degeneration and loss of anti-microbial peptide (AMP)-producing granules, indicative of malfunction of Paneth/stem cell network. Mechanistically, we found IL-22 upregulates ATF3, which is required to relay IL-22 signaling leading to STAT3 phosphorylation and subsequent AMP induction. Intriguingly, ATF3 itself does not act on STAT3 directly, instead ATF3 regulates pSTAT3 by negatively targeting protein tyrosine phosphatases (PTPs) including SHP2 and PTP-Meg2. Furthermore, we identified ATF3 is also involved in IL-6-mediated STAT3 activation in T cells and loss of ATF3 leads to reduced capacity of Th17 cells to produce their signature cytokine IL-22 and IL-17A. Collectively, our results suggest that via IL-22-pSTAT3 signaling in the epithelium and IL-6-pSTAT3 signaling in Th17 cells, ATF3 mediates a cross-regulation in the barrier to maintain mucosal homeostasis and immunity.

Dataset Information

Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

Publications

Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

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