An anti-cancer WxxxE-containing azurin polypeptide inhibits Rac1-dependent STAT3 and ERK/GSK-3β signaling in breast cancer cells

Abstract

In our previous study, we characterized a mycoplasmal small GTPase-like polypeptide of 240 amino acids that possesses an N-terminal WVLGE sequence. The N-terminal WVLGE sequence promotes activation of Rac1 and subsequent host cancer cell proliferation. To investigate the function of the WxxxE motif in the interaction with Rac1 and host tumor progression, we synthesized a 35-amino acid WVLGE-containing polypeptide derived from a cell-penetrating peptide derived from the azurin protein. We verified that the WVLGE-containing polypeptide targeted MCF-7 cells rather than MCF-10A cells. However, the WVLGE-containing polypeptide inhibited activation of Rac1 and induced cellular phenotypes that resulted from inhibition of Rac1. In addition, the WVLGE-containing polypeptide down-regulated phosphorylation of the STAT3 and ERK/GSK-3β signaling pathways, and this effect was abolished by either stimulation or inhibition of Rac1 activity. We also found that the WVLGE-containing polypeptide has a Rac1-dependent potential to suppress breast cancer growth in vitro and in vivo. We suggest that by acting as a Rac1 inhibitor, this novel polypeptide may be useful for the treatment of breast cancer.

Activation of STAT transcription factors by the Rho-family GTPases

“…The requirement for Rac1 was confirmed by co-expression with constitutively active Rac1, which overcame the peptide's inhibitory effect on STAT3 activation. Phenotypically, peptide-induced STAT3 inhibition decreased colony formation and reduced xenograft tumour growth [43], confirming that peptide inhibition of the Rac1-STAT3 axis could be of therapeutic utility.…”

“…Mutation of this motif abolished SGLP stimulated STAT3 activation by Rac1 and consequently blocked HeLa cell proliferation [42]. To investigate the therapeutic potential of the WxxxE motif, a 35mer peptide containing the WVLGE motif linked to an azurin cell penetrating peptide was synthesised [43]. This peptide could selectively target MCF7 cells over non-tumourigenic MCF-10A breast cancer cells.…”

SNHG6 Promotes Tumor Growth via Repression of P21 in Colorectal Cancer

“…Western blot analysis was performed as previously described [22]. Briefly, cells were lysed in cold lysis buffer, proteins (20-30μg) were resolved on SDS-PAGE, transferred onto PVDF membranes, and probed with antibodies to p21 (10355-1-AP, Proteintech), EZH2 (21800-1-AP, Proteintech), H3K27me (ab192985, Abcam) and GAPDH (sc-32233, Santa Cruz Biotechnology) at 4°C overnight.…”

EYA4 Promotes Cell Proliferation Through Downregulation of p27Kip1 in Glioma

“…Cells were lysed in cold lysis buffer as previously described [22], proteins (20-30μg) were resolved on SDS-PAGE, transferred onto PVDF membranes, and probed with antibodies for EYA4 (ab102648, Abcam, used dilution: 1:500), p27Kip1 (25614-1-AP, Proteintech, used dilution: 1:1000), Six1 (10709-1-AP, Proteintech, used dilution: 1:1000) and GAPDH (sc-32233, Santa Cruz Biotechnology, used dilution: 1:2000) at 4°C overnight. Detection was carried out with the SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit (Pierce, Rockford, IL, USA).…”