Cucurbitacin I Inhibits Rac1 Activation in Breast Cancer Cells by a Reactive Oxygen Species-Mediated Mechanism and Independently of Janus Tyrosine Kinase 2 and P-Rex1

Abstract

The small GTPase Rac1 has been widely implicated in mammary tumorigenesis and metastasis. Previous studies established that stimulation of ErbB receptors in breast cancer cells activates Rac1 and enhances motility via the Rac-guanine nucleotide exchange factor P-Rex1. As the Janus tyrosine kinase 2 (Jak2)/ signal transducer and activator of transcription 3 (Stat3) pathway has been shown to be functionally associated with ErbB receptors, we asked if this pathway could mediate P-Rex1/Rac1 activation in response to ErbB ligands. Here we found that the anticancer agent cucurbitacin I, a Jak2 inhibitor, reduced the activation of Rac1 and motility in response to the ErbB3 ligand heregulin in breast cancer cells. However, Rac1 activation was not affected by Jak2 or Stat3 RNA interference, suggesting that the effect of cucurbitacin I occurs through a Jak2-independent mechanism. Cucurbitacin I also failed to affect the activation of P-Rex1 by heregulin. Subsequent analysis revealed that cucurbitacin I strongly activates RhoA and the Rho effector Rho kinase (ROCK) in breast cancer cells and induces the formation of stress fibers. Interestingly, disruption of the RhoA-ROCK pathway prevented the inhibitory effect of cucurbitacin I on Rac1 activation by heregulin. Lastly, we found that RhoA activation by cucurbitacin I is mediated by reactive oxygen species (ROS). The ROS scavenger N-acetyl L-cysteine and the mitochondrial antioxidant Mito-TEMPO rescued the inhibitory effect of cucurbitacin I on Rac1 activation. In conclusion, these results indicate that ErbB-driven Rac1 activation in breast cancer cells proceeds independently of the Jak2 pathway. Moreover, they established that the inhibitory effect of cucurbitacin I on Rac1 activity involves the alteration of the balance between Rho and Rac.

Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

“…HRG and other ErbB ligands translocate P-Rex1 to the plasma membrane in a PI3K-dependent manner, leading to its activation. The requirement for P-Rex1 in HRG-induced Rac1 activation, ruffle formation, and motility, as well as its role in mammary tumorigenesis, has been unambiguously established by means of RNA interference (RNAi)-mediated loss of function approaches (36,40,43,44). Consistent with the established requirement for G␤␥ subunits in P-Rex1 activation, the GEF is also an effector of GPCRs (45).…”

“…Previously, we reported that treatment of breast cancer cells with HRG triggers a motile response that is mediated by the activation of Rac1 (35,36), a GTPase widely implicated in actin cytoskeleton reorganization, migration, and metastatic dissemination (37). Like most members of the Rho/Rac small G protein family, Rac1 is a molecular switch that cycles between inactive (GDP-bound) and active (GTP-bound) states.…”

Rho GTPases, oxidation, and cell redox control

“…The effects of cucurbitacin I on Rac1 and RhoA were dependent on intracellular ROS generation as treatment with the ROS scavenger NAC ablated the response due to cucurbitacin I exposure. 136 Yu et al characterized the time course of RhoA activity following H 2 O 2 stimulation in MDCK (Madin-Darby canine kidney) cells. They found a ~1.75-fold increase in RhoA-GTP levels that peaked approximately 5 min post stimulation.…”

Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture

“…In other words, 1 mg of CBI is equivalent to 112 mg of Bio 30 for therapy of cancers and many other PAK1-dependent diseases/disorders. This rough estimation is not far from the actual in vivo data where the daily dose of cucurbitacin B (1 mg/kg), closely related to CBI, and that of Bio 30 (50 mg/kg) are their effective dose to suppress the PAK1-dependent growth of pancreatic cancers or NF tumors in mice (14,20), suggesting that their in vivo bioavailability is quite similar.…”

“…In this study, we have chosen CBI and Bio 30 as potential model PAK1-blockers for the following reasons: CBI inhibits directly two activators (JAK2 and RAC) of PAK1 (19,20), while Bio 30 contains CAPE which down-regulates RAC by inhibiting directly AKR (AldoKeto-Reductase) 1B10 (21), and suppresses completely the PAK1-dependent growth of NF2-deficient tumor (schwannoma) in vivo (14).…”