Biochemical and Biophysical Research Communications
SHC1 sensitizes cancer cells to the 8-Cl-cAMP treatment
Introduction
Chemotherapeutic treatment with poor cancer cell specificity could lead to severe side effects for patients. Therefore, targeting cancer-specific cellular signal pathways is fundamental to the development of anti-cancer agents. 8-Chloro-cyclic AMP (8-Cl-cAMP) has been known to induce growth inhibition, apoptosis, reverse-transformation, and differentiation of cancer cells [1], [2], [3]; whereas, it has been shown to have no or minimal effects on non-transformed cells [4], [5]. Besides cyclic AMP-dependent protein kinase (PKA), 8-Cl-cAMP activates protein kinase C, Rap1 GTPase, Akt, AMP-activated protein kinase (AMPK), and p38 mitogen-activated protein kinase (p38 MAPK) during the induction of growth inhibition and apoptosis in cancer cells [4], [5], [6], [7], [8], [9]. The conversion of 8-Cl-cAMP to 8-Cl-adenosine, one of the metabolites of 8-Cl-cAMP, is essential to its anti-cancer activity [6], [8]. However, the accurate mechanisms pertinent to the cancer cell specificity of 8-Cl-cAMP have not been fully elucidated yet.
To search for signaling molecules regulating the activation of AMPK or p38 MAPK during the 8-Cl-cAMP-induced cancer cell growth inhibition, we performed mass spectrometric analysis of co-immunoprecipitated proteins by using anti-AMPK or p38 MAPK antibodies. Through this approach, we identified SHC1 (Src homology 2 domain-containing transforming protein 1) as one of the interacting partners of p38 MAPK.
SHC1 is an adapter protein containing Src homology 2 (SH2) domains that are conserved among cytoplasmic signaling proteins [10], [11]. SHC1 is involved in various receptor-mediated signaling pathways such as growth factors [12], [13], antigens [14], cytokines [15], [16], hormones [17], [18], and G-protein-coupled receptors [19]. Furthermore, SHC1 is highly phosphorylated and expressed in many types of transformed cells compared with their non-transformed counterparts [20], [21]. The cancer-specific induction and activation of SHC1 might regulate tumor progression, implying that SHC1 can be a good prognostic marker and therapeutic target [22], [23].
In this paper, we show that 8-Cl-cAMP and its metabolite, 8-Cl-adenosine, suppress SHC1 phosphorylation only in cancer cells, which is an upstream event of AMPK and p38 MAPK activation. We conclude that SHC1 is a crucial determinant of cancer-specific responsiveness to 8-Cl-cAMP.
Section snippets
Reagents and antibodies
8-Cl-cAMP was purchased from Biolog (Bremen, Germany). Compound C and ABT702 were from Calbiochem (San Diego, CA), and SB203580 was from A. G. Scientific (San Diego, CA). Phospho-SHC1 (Tyr239/240), phospho-AMPKα (Thr172), phospho-p38 MAPK (Thr180/Tyr182), phospho-MAPKAPK2 (Thr334), and total-MAPKAPK2 antibodies were purchased from Cell Signaling Technology (Danvers, MA). The β-actin antibody was purchased from Bioworld Technology (St. Louis Park, MN). Total-SHC1, total-p38 MAPK, total-AMPKα,
SHC1 interacts with p38 MAPK
We performed co-immunoprecipitation using anti-AMPK and anti-p38 MAPK antibodies to search for the signaling molecules participating in the AMPK and p38 MAPK pathways during the 8-Cl-cAMP-induced cancer cell growth inhibition. We then identified the proteins interacted with AMPK or p38 MAPK by using mass spectrometric analysis (Supplementary Table). Among them, SHC1 was selected for further study, because it interacts with various proteins as an adapter protein [24] and regulates diverse
Discussion
8-Cl-cAMP and its metabolite, 8-Cl-adenosine have been investigated as therapeutic agents against several cancers including solid tumors [32], multiple myeloma (ClinicalTrials.gov Identifier: NCT00004902), and leukemia (NCT00714103); however, the precise action mechanism of 8-Cl-cAMP's anti-tumor activity has not been fully identified. Previously, we showed that AMPK and p38 MAPK are essential factors for 8-Cl-cAMP-induced growth inhibition [8], [9]. In this study, we revealed that p38 MAPK
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A1A1002340, to Y.-H. Ahn) and the Ewha Womans University Research Grant of 2014 (1-2014-0714-001-1 to Y.-H. Ahn). K. Y. Choi and Y. J. Cho are supported by BK21 Research Fellowship from the Korean Ministry of Education, Science, and Technology.
References (33)
- et al.
Dual anticancer activity of 8-Cl-cAMP: inhibition of cell proliferation and induction of apoptotic cell death
Biochem. Biophys. Res. Commun.
(2000) - et al.
Reverse transformation of Harvey murine sarcoma virus-transformed NIH/3T3 cells by site-selective cyclic AMP analogs
J. Biol. Chem.
(1988) - et al.
Cooperative effect of 8-Cl-cAMP and rhGM-CSF on the differentiation of HL-60 human leukemia cells
Biochem. Biophys. Res. Commun.
(1991) - et al.
A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction
Cell
(1992) - et al.
A role for Src in signal relay by the platelet-derived growth factor alpha receptor
J. Biol. Chem.
(1998) - et al.
Evidence for a physical association between the Shc-PTB domain and the beta c chain of the granulocyte-macrophage colony-stimulating factor receptor
J. Biol. Chem.
(1996) - et al.
The Shc adaptor protein forms interdependent phosphotyrosine-mediated protein complexes in mast cells stimulated with interleukin 3
Blood
(2000) - et al.
Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity
Cell
(2001) - et al.
Impaired TrkB-mediated ERK1/2 activation in huntington disease knock-in striatal cells involves reduced p52/p46 Shc expression
J. Biol. Chem.
(2010) - et al.
The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein-protein interactions
Curr. Biol.
(1996)