A synthetic peptide hijacks the catalytic subunit of class I PI3K to suppress the growth of cancer cells

Highlights

• A peptide is designed to inhibit the activity of PI3K, a critical molecule for cell growth.

• The peptide is able reduce the proliferation of gastric cancer cells.

• The peptide can block tumor growth of gastric cancer cells in a mouse model.

• The peptide also inhibits the function of a hotspot mutation of PI3K found in many human cancers.

Abstract

Activation of class I Phosphoinositide 3-kinases (PI3Ks) by mutation or overexpression closely correlates with the development of various human cancers. Class I PI3Ks are heterodimers composed of p110 catalytic subunits and regulatory subunits represented by p85. PAQR3 has been found to inhibit p110α activity by blocking its interaction with p85. In this study, we identified the N-terminal 6–55 amino acid residues of PAQR3 being sufficient for its interaction with p110α. A synthetic peptide, P6-55, that contains the N-terminus of PAQR3 could disrupt the interactions of p110α with both PAQR3 and p85. The activity of PI3K was also inhibited by P6-55, accompanied by significant inhibition of cancer cell proliferation. In a xenograft mouse model, P6-55 was able to reduce tumor growth in vivo. Furthermore, P6-55 was capable of inhibiting the elevated basal PI3K activity of H1047R, a hotspot mutation found in many types of human cancers. The cell proliferation and migration of cancer cells bearing H1047R mutation were also reduced by P6-55. In conclusion, our study provides a proof of concept that blocking the interaction of p110α with p85 by a peptide can serve as a new strategy to inhibit the oncogenic activity of PI3K in cancer therapy.

Introduction

Phosphatidylinositol 3-Kinases (PI3Ks) belong to a family of lipid kinases that regulate multiple signaling pathways and control a plethora of physiological functions and cellular processes, including cell proliferation, growth, survival, motility, and metabolism. PI3Ks are divided into three classes (from class I to class III) based on their structures and substrate specificities [6], [25]. In mammals, class I PI3Ks are further divided into subclasses IA and IB, among which class IA PI3Ks are heterodimers composed of a p110 catalytic subunit and a regulatory subunit. The class IA p110 catalytic subunits are p110α, p110β, and p110δ encoded by genes PIK3CA, PIK3CB, and PIK3CD respectively. These three subunits associate with any of five regulatory isoforms including p85α (and its splicing variants p55α and p50α), p85β and p55γ. The class IB PI3Ks are heterodimers of a p110γ catalytic subunit (encoded by PIK3CG) in association with its regulatory subunits p101 or p87. While p110α and p110β are ubiquitously expressed, p110δ and p110γ are mainly restricted to leukocytes. In the absence of activating signals, p85 interacts with p110, leading to inhibition of p110 kinase activity. Upon activation by receptor tyrosine kinase (RTK) or G-protein coupled receptor (GPCR), class I PI3Ks are recruited to the plasma membrane, in which p85 inhibition of p110 is relieved, allowing p110 to phosphorylate PtdIns 4,5-bisphosphate (PtdIns(4,5)P2) and generate PtdIns(3,4,5)P3. This lipid product acts as a second messenger to activate AKT-dependent and AKT–independent downstream signaling pathways. On the other hand, the phosphatase and tensin homolog (PTEN) lipid phosphatase removes the 3′ phosphate from PtdIns(3,4,5)P3 to inactivate class I PI3K signaling pathway.

Frequent mutations of PIK3CA were firstly reported in human cancers in 2004 [23]. Later on, PI3K has come to the forefront as a major cancer-driver and potential drug target. In fact, PIK3CA is the second-most-frequently mutated oncogene, and PTEN is among one of the most-mutated tumor suppressor genes [6]. PIK3CA mutation has been firmly established as causative in many cancer types. Missense mutations occur in all domains of p110α, but the majority of mutations cluster in two hotspots, with the most common ones being E542K and E545K in the helical domain, and H1047R in the kinase domain. Cell-based studies identified that these hotspot mutations lead to cellular transformation via constitutive activation of p110α [11], [14], [38]. Several studies using genetically-engineered mouse models (GEMMs) also demonstrated the roles of mutant PIK3CA in tumor initiation, progression and maintenance [4], [15], [18], [31], [35]. Helical domain mutations reduce inhibition of p110α by p85 or facilitate direct interaction of p110α with insulin receptor substrate 1 (IRS1), while kinase domain mutations increase interaction of p110α with lipid membrane [2], [9], [22], [39]. Furthermore, it was found that the kinase domain mutation of p110α is dependent on the interaction with p85 [39].

As activating alterations in PI3K are highly frequent in a variety of human cancers and the importance of PI3K pathway in controlling many hallmarks of cancer development, PI3K has become a prime drug target for cancer therapy [6], [33]. Currently, there are three main classes of PI3K inhibitors in clinical testing, i.e., dual pan-PI3K/mTOR inhibitors, pan-PI3K inhibitors lacking significant mTOR activity and isoform-selective PI3K inhibitors. A major step forward in recent years is the introduction of over 30 small-molecule PI3K inhibitors into clinical trials and the first regulatory approval of the p110δ inhibitor idelalisib (also called CAL101) for multiple B-cell malignancies [36]. Due to the potential importance of PI3K pathway as a drug target, it is imperative to develop new types of PI3K inhibitors to alter the functionality of class I PI3K.

The progestin and adipoQ receptor family, named as PAQR, is a highly conserved protein family that is composed of eleven members: PAQR1 to PAQR11 [24]. PAQR3 was recently discovered as a new member of tumor suppressor that is deregulated in several types of human cancers including colon cancer, gastric cancer, hepatocellular carcinoma, bladder cancer, osteosarcoma, laryngeal squamous cell carcinoma, breast cancer, and prostate cancer [3], [10], [16], [17], [20], [26], [29], [30], [32], [34]. PAQR3, also named RKTG for Raf kinase trapping to Golgi, is a seven-transmembrane protein specifically localized in the Golgi apparatus in mammalian cells [5], [19]. PAQR3 has been discovered to be a negative regulator of Raf-1 by sequestrating Raf-1 to the Golgi apparatus, thereby blocking Ras-Raf-MEK-ERK signaling pathway [5], [19]. PAQR3 has a negative role in the regulation of angiogenesis of endothelial cells [37]. PAQR3 functionally interacts with p53 in cancer formation and plays an important role in epithelial-mesenchymal transition (EMT) and tumor metastasis [8], [12]. PAQR3 also inhibits AKT activation by two mechanisms: by inhibiting signaling of G protein βγ-subunit and by inhibiting PI3K via spatial regulation of p110α subunit [13], [27]. In this study, we further explored the inhibitory activity of PAQR3 on PI3K signaling and identified the minimal domain of PAQR3 required for the inhibition. Consequently, we designed a synthetic peptide to mimic the inhibitory activity of PAQR3 and analyzed its effect on tumor growth both in vitro and in vitro using a human gastric cancer model.