Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner

Abstract

BackgroundSmurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. The purpose of the present study is to examine the effect of Smurf2 knockdown on the tumorigenic potential of human breast cancer cells emphasizing more on proliferative signaling pathway.MethodssiRNAs targeting different regions of the Smurf2 mRNA were employed to knockdown the expression of Smurf2. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth, cell cycle arrest, and cell cycle and cell proliferation related protein expressions upon Smurf2 silencing.ResultsSmurf2 silencing in human breast cancer cells resulted in a decreased focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of Smurf2 suppressed cell proliferation. Cell cycle analysis showed that the anti-proliferative effect of Smurf2 siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by decreased expression of cyclin D1and cdk4, followed by upregulation p21 and p27. Furthermore, we demonstrated that silencing of Smurf2 downregulated the proliferation of breast cancer cells by modulating the PI3K- PTEN-AKT-FoxO3a pathway via the scaffold protein CNKSR2 which is involved in RAS-dependent signaling pathways. The present study provides the first evidence that silencing Smurf2 using synthetic siRNAs can regulate the tumorigenic properties of human breast cancer cells in a CNKSR2 dependent manner.ConclusionsOur results therefore suggest a novel relation between Smurf2 and CNKSR2 thereby regulating AKT-dependent cell proliferation and invasion. Owing to the fact that PI3K-AKT signaling is hyperactivated in various human cancers and that Smurf2 also regulates cellular transformation, our results indicate that Smurf2 may serve as a potential molecule for targeted cancer therapy of certain tumour types including breast cancer.

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes

“…While SMURF2 and HACE1 have been implicated in cell migration by RNA interference studies (Castillo-Lluva et al, 2013; David et al, 2014; Jin et al, 2009), we wondered whether our unprecedented ability to activate (or block) enzyme activity with UbVs could both score these positive controls and also potentially reveal roles for other HECT E3s not known previously as regulators of this pathway. To this end, we transduced HCT116 human colon cancer carcinoma cells with a pool of 83 distinct lentiviruses, each containing an inducible defined UbV expression cassette targeting one of 19 HECT E3s or one of 13 other proteins, and analyzed the migratory response in a trans-well migration assay by deep sequencing (Figure 7A–B).…”

Disease-associated synaptic scaffold protein CNK2 modulates PSD size and influences localisation of the regulatory kinase TNIK

“…Among the known CNK proteins, mammalian CNK2 isoforms are the proteins that exhibit tissue-specific expression in nervous tissue 9 . There are studies on how CNK2 isoforms participate in protein-protein interactions [9][10][11] , but relatively few studies focus specifically on the scaffold function of CNK2 in neurons and in the brain, despite an increasing number of genetic studies that directly implicate CNK2 mutations in human brain disorders. Specifically, both deletions [12][13][14][15] and point mutations [16][17][18][19] that result in loss of function of CNK2 cause X-linked intellectual disability (XLID) that is typically associated with seizures.…”

ImageJ macros for the user-friendly analysis of soft-agar and wound-healing assays

“…Moreover, they are easy to use, require minimal user input, and present numerous advantages relative to other methods currently in use. Additionally, these macros are able to perform large-scale automated analyses of an infinite amount of images, which is not possible using some methods published to date (17,19,24,25), These macros also minimize manual intervention and eliminate the need for expensive and robust fluorescence microscopy tools, or commercial software for image processing, such as Image-Pro Plus (13). A previous study compared the use of Matlab and ImageJ for assessing the migration rate from an in vitro circular model.…”