PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF,P-Rex1

The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers.The phosphoinositide 3-kinase (PI3K) family of lipid kinases plays a prominent role in the growth and survival of several types of cancer (1). The PI3K pathway is aberrantly activated by a number of different mechanisms in cancers. These include genetic mutation and/or amplification of key pathway components, such as amplification or mutation of the PI3K catalytic subunit p110α (encoded by PIK3CA gene), mutation or deletion of the phosphatase PTEN, amplification or mutation of the gene encoding for the PI3K effector protein kinase AKT, as well as constitutive activation of receptor tyrosine kinases (RTKs) (e.g., HER2 amplification in breast cancer) or other less frequent events (2). PI3K phosphorylates the phosphoinositide PI(4,5)P2 in the 3′OH group of the inositol ring to produce PI(3,4,5)P3. PI(3,4,5)P3 directly binds to the pleckstrin homology (PH) domains of certain proteins, such as AKT, leading to their activation, which in turn transmit growth and survival signals.These findings have encouraged the development of several different PI3K inhibitors, many of which are either in or approaching clinical trial testing. Genotype-driven patient selection has been investigated to uncover patient populations that will be particularly susceptible to PI3K inhibitors. Cancers harboring mutations in the PIK3CA gene have emerged as among the most sensitive to single-agent PI3K inhibitors in several preclinical studies, although clinical activity to date has been mixed (3–6). These gain-of-function mutations in the PI3KCA gene are found in a broad range of cancers, and they are highly enriched in breast cancer, where they are observed in 20–25% of cases (7). In addition, breast cancers with amplified HER2, which comprise ∼20% of all breast cancers, (8) are also particularly sensitive to PI3K inhibition (9–11). However, even among patients whose cancers harbor PIK3CA mutations, a significant heterogeneity of responses has been observed to PI3K inhibitors currently being tested in clinical studies (3–5). There have been some patients with bona fide response evaluation criteria in solid tumors (RECIST) criteria responses, but the majority has not had similarly impressive outcomes. These early clinical results highlight the potential utility of a biomarker of sensitivity to single-agent PI3K inhibitors.Interestingly, early clinical trial reports have found that inhibition of PI3K signaling may sometimes lead to suppression of protein kinase MEK (MEK)/ERK signaling (6). Although a previous laboratory study had shown that the PI3K/mammalian target of rapamycin (mTOR) inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin can inhibit protein kinase RAF (RAF)/MEK/ERK-signaling (12), this clinical observation was initially surprising because several studies have shown that inhibitors of components of the PI3K signaling pathway (such as AKT and mTOR inhibitors) actually lead to activation of the MEK/ERK signaling in many cancer types (11, 13), and such feedback activation may impair sensitivity to PI3K pathway inhibitors (9, 11, 14). Because PIK3CA and HER2 amplified breast cancers are particularly sensitive to single-agent PI3K pathway inhibitors, we investigated how PI3K inhibitors impact MEK/ERK signaling in these genetically defined subsets of breast cancers. In our study, we found that several cell lines harboring PIK3CA mutation and/or HER2 amplification suppress MEK/ERK pathway signaling as well as the AKT pathway after treatment with PI3K inhibitors, and importantly, inhibition of both pathways is necessary for maximal antitumoral activity. Moreover we identify that the mechanistic link between PI3K and MEK/ERK is via a PI(3,4,5)P3-dependent regulation of the phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1)/ small GTPase Rac1 (Rac1)/protein kinase c-RAF (c-RAF) pathway in these cancers. Importantly, the expression levels of the Rac guanine exchange factor (Rac-GEF), P-Rex1, correlate with sensitivity to PI3K inhibitors in these breast cancer cell lines.