Both TGF-β and the PI3K-AKT signaling pathways are known activators of various intracellular pathways that regulate critical cellular functions, including cancer cell survival and proliferation. The interplay between these two oncogenic pathways plays a major role in promoting the initiation, growth, and progression of tumors, including breast cancers. The molecular underpinning of the inter-relationship between these pathways is, however, not fully understood, as is the role of WAVE3 phosphorylation in the regulation of tumor growth and progression. WAVE3 has been established as a major driver of the invasion–metastasis cascade in breast cancer and other tumors of epithelial origin. WAVE3 phosphorylation downstream of PI3K was also shown to regulate cell migration. Here we show that, in addition to PI3K, WAVE3 tyrosine phosphorylation can also be achieved downstream of TGF-β and EGF and that WAVE3 tyrosine phosphorylation is required for its oncogenic activity. Our in vitro analyses found loss of WAVE3 phosphorylation to significantly inhibit cell migration, as well as tumorsphere growth and invasion. In mouse models for breast cancer, loss of WAVE3 phosphorylation inhibited tumor growth of two aggressive breast cancer cell lines of triple-negative subtype. More importantly, we found that WAVE3 phosphorylation is also required for the activation of PI3K, TGF-β, and EGF signaling and their respective downstream effectors. Therefore, our study identified a novel function for WAVE3 in the regulation of breast cancer development and progression through the modulation of a positive feedback loop between WAVE3 and PI3K-TGF-β-EGF signaling pathways.

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Post-translational modification of proteins, such as tyrosine phosphorylation, plays a major role in driving the oncogenic activity of oncogenes. WAVE3 (WASF3), an adaptor and actin cytoskeleton remodeling protein, contributes to cell migration, cancer cell invasion, and metastasis. WAVE3 plays a vital role in the progression and metastasis of triple negative breast cancer (TNBC), in part through the regulation of cancer stem cells (CSCs). Several studies have shown that WAVE3 tyrosine phosphorylation is required for its oncogenic activity. Moreover, our recent study showed that the proline rich domain (PRD) of WAVE3 is required for maintenance of the CSC niche in breast cancer by regulating the nuclear translocation of the CSC-specific nuclear transcription factor YB1. Here, we show that the PRD domain of WAVE3 and its phosphorylation are essential for driving the oncogenic activity of WAVE3. We show that phosphorylation of WAVE3 PRD is essential for migration and invasion of breast cancer cells in vitro, as well as tumor growth and metastasis in vivo. Mechanistically, we show that phosphorylation of the WAVE3 PRD is essential for interaction between WAVE3 and YB1. Loss of PRD phosphorylation inhibits such interaction and the YB1-mediated activation of expression of CSC markers, as well as the WAVE3 mediated activation of EMT. Together, our study identifies a novel role of WAVE3 and its PRD domain in the regulation of the invasion metastasis cascade in BC that is independent of the known function of WAVE3 as an actin cytoskeleton remodeling protein through the WAVE regulatory complex (WRC).

Nerve agents, the deadliest chemical warfare agents, are potent inhibitors of acetylcholinesterase (AChE) and cause rapid cholinergic crisis with serious symptoms of poisoning. Oxime reactivators of AChE are used in medical practice in the treatment of nerve agent poisoning, but the search for novel improved reactivators with central activity is an ongoing pursuit. For numerous oximes synthesized, in vitro reactivation is a standard approach in biological evaluation with little attention given to the pharmacokinetic properties of the compounds. This study reports a comprehensive physicochemical, pharmacokinetic, and safety profiling of five lipophilic 3-hydroxy-2-pyridine aldoximes, which were recently shown to be potent AChE reactivators with a potential to be centrally active. The oxime JR595 was singled out as highly metabolically stable in human liver microsomes, noncytotoxic oxime for SH-SY5Y neuroblastoma and 1321N1 astrocytoma cell lines, and its pharmacokinetic profile was determined after intramuscular administration in mice. JR595 was rapidly absorbed into blood after 15 min with simultaneous distribution to the brain at up to about 40% of its blood concentration; however, it was eliminated from both the brain and blood within an hour. In addition, the MDCKII-MDR1 cell line assay showed that oxime JR595 was not a P-glycoprotein efflux pump substrate. Finally, the preliminary antidotal study against multiple LD 50 doses of VX and sarin in mice showed the potential of JR595 to provide desirable therapeutic outcomes with future improvements in its circulation time.