Blocking Tumor Cell Migration and Invasion with Biphenyl Isoxazole Derivative KRIBB3, a Synthetic Molecule That Inhibits Hsp27 Phosphorylation

Abstract

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC 50 ‫؍‬ 5 and 50 nM, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has antimigratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.Traditionally, genetic mutagenesis has proven to be a useful tool in solving the function of a wide range of genes in biological process. Recently, a chemical genetic approach has been developed to elucidate the principles of a wide range of biological processes (for review, see Refs. 1-3). In chemical genetics, instead of site-specific mutation as in traditional genetics, the function of a protein is inhibited or activated using small chemicals. Therefore, chemical genetics seeks to identify novel small molecules that afford functional dissection of cell biological pathways. Such chemicals are useful as bioactive molecular probes and allow further analysis of the relationship between target processes or proteins within cells and their cellular function.Metastasis plays a major role in morbidity and mortality from breast cancer (4). The metastatic potential of cancer cells is related to the ability to digest the extracellular matrix, migrate, cross blood vessel walls, and reach the blood circulation (for review, see Refs. 5-8). Cell movement is a complex process involving a number of steps, including the disruption of cell-cell junctions, cytoskeletal rearrangements, and the constant remodeling of adhesive contacts with the extracellular matrix (for review, see Refs. 9 -11). Cell migration contributes to several other important pathological pro...

p90 ribosomal S6 kinase 2 promotes invasion and metastasis of human head and neck squamous cell carcinoma cells

“…Hsp27 activity is regulated by phosphorylation at Ser15, Ser78, and Ser82, which induces redistribution of the large oligomers into small tetrameric units (38) and Hsp27 nuclear translocation to prevent apoptosis (39). Recently, it was reported that attenuation of Hsp27 phosphorylation by the specific microtubule inhibitor KRIBB3 results in inhibition of tumor cell migration and invasion (25), while enhanced phosphorylation at Ser78 of Hsp27 significantly correlates with HER-2/neu and LN positivity in breast cancer (24). Our findings demonstrate that RSK2 signals through Hsp27 to regulate actin filaments and cell invasion via direct phosphorylation of Hsp27 at both Ser78 and Ser82 but not …”

“…Hsp27 is regulated by phosphorylation at Ser15, Ser78, and Ser82. Phosphorylation of Hsp27 is associated with tumor cell migration and invasion and correlates with LN positivity in breast cancer (24,25). We first determined whether RSK2 directly phosphorylates Hsp27 in an in vitro kinase assay, in which purified recombinant Hsp27 (rHsp27) WT and individual S15A, S78A, or S82A mutant proteins were incubated with active recombinant RSK2 (rRSK2).…”

Hsp-27 expression at diagnosis predicts poor clinical outcome in prostate cancer independent of ETS-gene rearrangement

“…Inhibition of HSPB1 expression modulated apoptosis and abrogated the malignant phenotype of human prostate cancer cells, thus identifying Hsp-27 as a potential therapeutic target. Separately, the biphenyl isoxasole KRIBB3 inhibits tumour cell migration by blocking protein kinase C-dependent phosphorylation of Hsp27 (Shin et al, 2005) to induce mitotic arrest and to enhance apoptosis (Shin et al, 2008). Recently, it has been shown that pyrrolo-pyrimidones, a novel class of p38 MAPK/MAPK-activated protein kinase 2 (MK2) inhibitors, inhibit phosphorylation of Hsp-27, its downstream target (Schlapbach et al, 2008), and that inhibition of Hsp-27 phosphorylation at Ser 78 and Ser 82 by the MAPKAP kinase MK5 prevents the F-actin reorganisation that is necessary for cell migration (Kostenko et al, 2009).…”

KRIBB11 Inhibits HSP70 Synthesis through Inhibition of Heat Shock Factor 1 Function by Impairing the Recruitment of Positive Transcription Elongation Factor b to the hsp70 Promoter

“…Western Blotting-Lysates were prepared using RIPA buffer as described previously (41). A volume of lysate corresponding to 30 g was subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore).…”