Elsevier

Surgery

Volume 154, Issue 2, August 2013, Pages 404-410
Surgery

Society of University Surgeons
An MAPK-dependent pathway induces epithelial-mesenchymal transition via Twist activation in human breast cancer cell lines

Presented at the Academic Surgical Congress 2013, New Orleans, LA, February 5–7, 2013.
https://doi.org/10.1016/j.surg.2013.05.012Get rights and content

Background

Twist is an epithelial-mesenchymal transition (EMT) transcription factor that instigates cell invasion. Our research has shown that osteopontin (OPN) regulates the EMT factor Twist. The underlying signaling pathway is unknown. We hypothesized that OPN activates Twist to induce EMT in human breast cancer.

Methods

Potential kinases for Twist were identified using NetPhosK. Inhibitors of MEK1/2, JNK, p38 MAPK, and PI3K were applied to human breast cancer cells MDA-MB231 (OPN high). After 24 h, Twist was immunoprecipitated and incubated with phosphoserine. Expression of the Twist target protein, Bmi-1, was determined following 24-h osteopontin aptamer (APT) treatment; mutant aptamer (MuAPT) was used as the control. Scratch-wound assay was imaged 12, 24, and 48 h after APT and MuAPT treatment.

Results

MEK1/2 inhibition caused ∼twofold decrease in Twist serine phosphorylation (P < .05). APT blockade of OPN in MB231 decreased Bmi1 protein twofold (P < .05). Aptamer-treated cells were significantly decreased in cell migration and wound closure in the scratch wound-assay (P < .001).

Conclusion

We demonstrate that OPN extracellular binding to MB231 activates an autocrine MAPK intracellular signaling pathway resulting in Twist activation and promoting Bmi1 expression to further EMT initiation and cellular migration. Our results elucidate a previously undescribed role for OPN as a prime regulator of EMT in human breast cancer cells.

Section snippets

RNA aptamer

The osteopontin-R3 aptamer, mutant, and all test variants were synthesized by Dharmaco (Lafayette, CO). Synthesized osteopontin-R3 and its mutant osteopontin-R3 with 2′-O-methylated purines, 5′ cholesterol, and 3′-IDT modification were used for in vitro studies. This aptamer has the usual stem-loop structure of other RNA aptamers. Osteopontin-R3 had been tested previously for affinity and specificity for osteopontin by using RNA electrophoretic mobility shift assays and its in vitro half-life

MEK1/2 inhibition decreased Twist serine phosphorylation in MB-231

The potential kinases targeting Twist were identified by sequence analysis using NetPhosK. In MB-231 control, the relative band density of Twist serine phosphorylation standardized to beta actin was 1.30. Among the kinase inhibitions, MEK1/2 inhibition caused a significant decrease in Twist serine phosphorylation (Fig 1, A) with a relative band density of 0.61 (P < .05). The remaining kinase inhibitions decreased Twist serine phosphorylation but not to the extent of MEK1/2 inhibition. The

Discussion

Osteopontin is upregulated in a variety of metastatic tumors; it plays a substantial role in tumor invasion, migration, and differentiation. In this study we built on previous findings that osteopontin mediates the serine phosphorylation of Twist, an EMT-TF. We found that inhibition of MEK1/2 kinase, a component of the MAPK cascade, causes Twist serine phosphorylation to decrease compared to MB-231 control and other kinase inhibitors. When inhibiting osteopontin with aptamer, which decreases

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