We have previously reported that expression of SIRPalpha1/SHPS-1 was strongly suppressed in v-Src-transformed cells and its forced expression resulted in the suppression of anchorage-independent growth of the cells [K. Machida, S. Matsuda, K. Yamaki, T. Senga, A.A. Thant, H. Kurata, K. Miyazaki, K. Hayashi, T. Okuda, T. Kitamura, T. Hayakawa, M. Hamaguchi, v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells, Oncogene 19 (2000) 1710-1718]. We examined the effect of human SIRPalpha1 expression in breast cancer cell lines, Hs578T and MCF7, and compared with the effect of SIRPalpha2 expression in Hs578T. Forced expression of either SIRPalpha1 or SIRPalpha2 did not perturb the growth of Hs578T in a conventional attached condition. Their expression, however, enforced the actin stress fiber formation and induced activation of Rho, but not Rac, in Hs578T cells. Moreover, forced expression of either SIRPalpha1 or SIRPalpha2 displayed distinct suppressive effect on the anchorage-independent growth of Hs578T cells. Similarly, forced expression of SIRPalpha1 in MCF7 specifically suppressed the anchorage-independent growth of the cells. Taken together, our results strongly suggest the function of SIRPalpha1 and 2 as type II tumor suppressors for human breast carcinoma.
"To establish MDA-MB-231  and MCF10A cell lines that constitutively expressed GFP, GFP-wt (GFP-tagged wild-type palladin) and GFP-S77.197G (GFP-tagged mutant palladin), each cDNA was cloned into a pQCXIN retrovirus vector (Clontech, Mountain View, CA). "
[Show abstract][Hide abstract] ABSTRACT: Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signal-regulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of non-phosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration.
PLoS ONE 12/2011; 6(12):e29338. DOI:10.1371/journal.pone.0029338 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cutaneous melanoma is the most aggressive skin cancer; it is highly metastatic and responds poorly to current therapies. The expression of platelet-derived growth factor receptors (PDGF-Rs) is reported to be reduced in metastatic melanoma compared with benign nevi or normal skin; we then hypothesized that PDGF-Ralpha may control growth of melanoma cells. We show here that melanoma cells overexpressing PDGF-Ralpha respond to serum with a significantly lower proliferation compared with that of controls. Apoptosis, cell cycle arrest, pRb dephosphorylation, and DNA synthesis inhibition were also observed in cells overexpressing PDGF-Ralpha. Proliferation was rescued by PDGF-Ralpha inhibitors, allowing to exclude nonspecific toxic effects and indicating that PDGF-Ralpha mediates autocrine antiproliferation signals in melanoma cells. Accordingly, PDGF-Ralpha was found to mediate staurosporine cytotoxicity. A protein array-based analysis of the mitogen-activated protein kinase pathway revealed that melanoma cells overexpressing PDGF-Ralpha show a strong reduction of c-Jun phosphorylated in serine 63 and of protein phosphatase 2A/Balpha and a marked increase of p38gamma, mitogen-activated protein kinase kinase 3, and signal regulatory protein alpha1 protein expression. In a mouse model of primary melanoma growth, infection with the Ad-vector overexpressing PDGF-Ralpha reached a significant 70% inhibition of primary melanoma growth (P < .001) and a similar inhibition of tumor angiogenesis. All together, these data demonstrate that PDGF-Ralpha strongly impairs melanoma growth likely through autocrine mechanisms and indicate a novel endogenous mechanism involved in melanoma control.
Neoplasia (New York, N.Y.) 09/2009; 11(8):732-42. DOI:10.1593/neo.09408 · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Normal mammary development requires coordinated interactions of numerous factors, including prolactin (PRL) and insulin-like growth factor I (IGF-I), both of which have also been implicated in breast cancer pathogenesis and progression. We previously reported that PRL and IGF-I synergize in breast cancer cells to activate ERK1/2 and AKT, leading to increased proliferation, survival, and invasion. Intriguingly, PRL co-treatment with IGF-I augments IGF-I receptor (IGF-IR) phosphorylation 2-fold higher than IGF-I alone. Here, we showed the importance of the tyrosine phosphatase SHP-2 in this cross-talk using pharmacological inhibition and small interfering RNA. SHP-2 recruitment to IGF-IR was significantly attenuated by PRL co-treatment. Src family kinase activity was required for IGF-IR association with SHP-2, ligand-induced IGF-IR internalization, and PRL-enhanced IGF-IR phosphorylation. Inhibition of internalization, via knockdown of the GTPase, dynamin-2, prevented not only IGF-IR dephosphorylation, but also PRL-enhanced IGF-IR phosphorylation. Consistently, PRL diminished IGF-I-induced IGF-IR internalization, which may result from reduced SHP-2 association with IGF-IR, because we demonstrated an essential role for SHP-2 in IGF-IR internalization. Together, these findings describe a novel mechanism of cross-talk between PRL and IGF-I in breast cancer cells, with implications for our understanding of tumor progression and potential therapeutic strategies.
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