The phosphatidylinositol 3-kinases (PI3Ks) are a group of highly conserved lipid kinases with important roles in determining cellular fate. Although they are associated with the protein kinases that phosphorylate proteins, PI3Ks phosphorylate lipids by adding a phosphate group, thereby converting phosphatidylinositol-4, 5-triphosphate (PIP2) to phosphatidylinositol-3, 4,5-triphosphate (PIP3), the second messenger that recruits AKT to the plasma membrane through its interaction with the pleckstrin-homology domain of AKT [2]. Subsequently, downstream pathways, including the mechanistic target of rapamycin 1 (mTORC1) pathway, are activated. PI3K can be activated by receptor tyrosine kinases (RTKs), cytokine/chemokine receptors, G-protein coupled receptors and B/T-cell receptors. Therefore, the PI3K pathway regulates cell growth, nutrition and metabolism, cytoskeletal remodeling, cell proliferation and even cell death [3], thereby controlling normal cell physiology and cellular transformation.
The PI3K family is divided into three classes (classes 1–3) according to structural differences. Class 1 is the most widely studied and is further subcategorized into 1A and 1B subtypes, which transduce RTK and G-protein-coupled-receptor signaling, respectively. Type 1A PI3Ks are heterodimers consisting of a catalytic subunit (p110) and a regulatory subunit (p85 or p55). Four distinct genes encode PI3K catalytic subunits, p110α/β/γ/δ, whereas the regulatory subunit is encoded by three genes that are translated into p85α, p85β, p55γ and splice variants. In basal state or in the absence of stimuli, the p85 subunit binds the p110 subunit and inhibits its catalytic activity [4]. After stimulation, such as by growth factors, the p110/p85 complex is recruited to the plasma membrane, where p85, via its SH2 domains, binds tyrosine-phosphorylated RTKs or adaptor proteins, such as insulin receptor substrate 1 (IRS1) [5], thus leading to the displacement of p85 from the PI3K complex and subsequent p110 activation. Hence, the expression level of p85 inside cells is critical in regulating the activation of p110. In parallel, cells have evolved a checkpoint system to turn off or limit PI3K-AKT activation, in which several lipid phosphatases, particularly phosphatase and tensin homolog (PTEN), convert PIP3 back to PIP2 by removing its 3´-phosphate [2].
The link between the PI3K pathway and cancer can be traced back to the original discovery of the first PI3K activity associated with oncogene-induced transformation, for instance, by Src and polyomavirus middle T antigen [6]. PIK3CA, which encodes the p110α catalytic subunit of PI3K, is among the most frequently mutated oncogenes in human cancers [7]. The negative regulator, the phosphatase PTEN, is also frequently mutationally inactivated in tumors; consequently, the normal kinase/phosphatase balance is disrupted, thus resulting in constitutive elevation of PIP 3 and activation of the PI3K-AKT pathway driving transformation and tumor progression [8]. While the p85α subunit is considered a tumor suppressor, the p85β subunit actually promotes tumor development [9]. In various cancers, PIK3R1, the gene encoding p85α, contains mutations that disrupt its binding with p110. In contrast, PIK3R2, the gene encoding p85β, is overexpressed in tumors [9]. Furthermore, p85 functions in a p110-independent manner, largely through acting as an adaptor protein that facilitates processes including signal transduction regulating the insulin response, senescence, unfolded-protein stress, cytoskeletal rearrangement and endocytic trafficking [10]. However, understanding of exactly how these PI3K isoforms regulate tumor progression remains incomplete.
The authors have previously reported that p110α helical-domain hotspot mutations lead to direct association of the protein with IRS1, independently of p85, thus resulting in recruitment of the mutant p110α to the plasma membrane, conversion of PIP2 to PIP3 and promotion of tumor growth [11]. However, tumors with PIK3CA helical-domain mutations rather than hotspot mutations in the catalytic domain are believed to be less responsive to inhibitors targeting PI3K, AKT or mTOR [12]. These results have also suggested the existence of yet-unidentified mechanisms driving the distinct phenotypes of the p110 helical-domain mutants. In the current study, Hao et al. have reported an unexpected, intriguing observation regarding the role of the p85β, but not the p85α subunit, in specifically promoting tumor development in cancer cells with p110 helical-domain mutations, but not wild-type or kinase-domain-mutant p110. They first demonstrated a diminished interaction between the p85β, but not the p85α subunit, and p110 with helical-domain mutations, but not wild-type or kinase-domain-mutant p110. By aligning the amino acid sequences of p85β and p85α, the authors found that the N-terminal regions of the two isoforms are less highly conserved than the C-terminal regions containing the two SH2 domains. By swapping the N-terminal regions, which include the SH3 and GAP domains, between p85β and p85α, they confirmed that this region of p85β is indeed responsible for the lower affinity of p85β toward p110, thus leading to higher levels of the free form of p85β.
To determine the importance of p85β in cancer, the authors analyzed TCGA data in colon, bladder, endometrial and breast cancers, and found that high expression of PIK3R2 (encoding p85β) correlates with poorer prognosis only in cancers bearing p110 helical-domain mutations, thereby indicating a tumor-promoting role of p85β. Consistently, they observed that knockdown of p85β specifically inhibits the growth of tumors derived from colon cancer cells with p110 helical-domain mutations, yet has no effect on tumors from cancer cells with wild-type or kinase-domain-mutant p110.
This result prompts an important question regarding how p85β promotes the growth of tumors bearing p110 helical-domain mutations. The authors ruled out the involvement of canonical PI3K downstream factors such as AKT and mTOR in this process, thus suggesting a previously unknown mechanism. During their analysis, the authors observed an increase in the nuclear signal of p85β in cancer cells expressing the p110 helical-domain mutations, thereby suggesting that free p85β can translocate into the nucleus. They indeed found a non-canonical nuclear localization signal (NLS, amino acids 474–484 with a sequence of LQMKRTAIEAF) in p85β that mediates its nuclear translocation.
To understand how nuclear p85β drives tumor progression, the authors conducted RNA sequencing in isogenic cell lines expressing wild-type p85β or an NLS-disrupted mutant (K477R478 to A477A478). The results revealed that many tumor-suppressing genes, including ATM, BRCA1/2 and APC, were upregulated in p85β NLS-mutant cells, indicating that wild-type p85β in the nucleus downregulates tumor-suppressor genes, thus potentially contributing to its tumor-promoting role. Further analysis indicated that p85β specifically upregulated the level of trimethylated H3K27, but not that of H3K9, H3K36 or H3K76, thus resulting in global gene transcriptional regulation. They then found that p85β increased the protein levels of the EZH1/2 methyltransferases, but not the other subunits of the PRC2 repressor complex. Although p85β acts a global transcriptional regulator, its effect on EZH1/2 levels occurred not through transcriptional upregulation but through protein-stability regulation. Again, these changes were observed only in cancer cells with p110 helical-domain mutations. The authors then demonstrated that p85β stabilized EZH1/2 through recruiting the deubiquitinase USP7 (also called HAUSP), which removed ubiquitin from EZH1/2 and led to the stabilization of these two histone methyltransferases.
The authors further tested whether combining an EZH1/2 inhibitor with a PI3K inhibitor might specifically target colon cancers with p110 helical-domain mutations. As expected, the combination treatment suppressed the xenograft tumor growth of human colon cancer cell lines bearing helical-domain mutations of p110 more strongly than that of cell lines with kinase-domain-mutant or wild-type p110, as confirmed in colorectal PDX tumors with p110 helical-domain mutation.
In conclusion, Hao et al. [1] have presented compelling and exciting findings regarding a novel function of p85β in the nucleus, thus filling a knowledge gap in the understanding of how the PI3K pathway drives cancer progression specifically in the genetic background of p110 helical-domain mutations ( Figure 1 ). These findings also suggest an effective approach to treating tumors with p110α helical-domain mutations by combining an EZH1/2 inhibitor with a PI3K inhibitor.
Model of the novel nuclear function of p85β in promoting tumor growth.
Receptor tyrosine kinase (RTK) activates the p110/p85 complex, which stimulates cell growth through two routes. p85β binds wild-type (WT) or kinase-domain-mutant p110, forming the canonical PI3K complex, which then activates downstream AKT and mTOR, and promotes gene expression and cell growth. However, when p110 has mutations in the helical domain (mH), p85β disassociates from p110 and translocates into the nucleus though its nuclear localization signal. In the nucleus, p85β recruits USP7 to EZH1/2; consequently, the stabilization of EZH1/2 increases H3K27 trimethylation and global transcriptional gene expression.
Although these findings offer innovative insights into PI3K signaling and its effects on tumor progression, they also prompt several questions worthy of further investigation. First, why does p85β demonstrate these effects only in cancer cells with helical-domain-mutant p110? For nuclear translocation, is the binding affinity between p85β and the helical-domain-mutant p110 greatly diminished? X-ray crystallography and cryo-EM studies have shown that the N-terminal ABD domain of p110α binds the internal nSH2 and iSH2 domains of p85α [13, 14]. However, the ABD domain in p110 and the SH3 domain in p85, which are considered to interact with each other, tend to be flexible and therefore have failed to produce high resolution structures. Yet, the finding that the addition of a specific p110 inhibitor stabilizes the structures of these domains suggests that the N-terminal SH3 domain of p85α, which was also responsible for the observed differences in p110 binding in the current study, points toward the catalytic domain but not the helical domain of p110α [13]. Hence, whether the amino terminus of p85β binds the helical domain of p110 remains unknown. Future structural studies will be important to answer these questions. The identified NLS (LQMKRTAIEAF) in p85β meets only the minimum requirement (i.e., KR) for an NLS; hence, whether post-translational modifications such as phosphorylation on T479, methylation on R478 or acetylation on K477 might be involved in promoting p85β nuclear shuttling is an interesting question worthy of investigation. In addition, why p85β does not interact with USP7 or EZH1/2 in the nucleus in non-helical p110-mutant cancer cells is unknown, given that the p110 subunit should be irrelevant to the function of p85β in the nucleus. Additionally, USP7 is a widely studied de-ubiquitin enzyme that regulates tumors through stabilizing many important proteins that drive/promote or suppress tumor progression, such as CHK1, MDM2, c-MYC, P53 and PTEN, probably in a context dependent manner [15]. This study adds another protein, EZH1/2, to the long list of USP7 substrates that promote colon tumors. Hence, combining a USP7 inhibitor with a PI3K inhibitor may produce a similar anti-tumor effect to that seen after treatment with the combination of EZH1/2 inhibitor and PI3K inhibitor reported here. Finally, if the nuclear signaling is specific to the helical-domain mutations of p110, an inhibitor that can specifically target helical-domain mutations without acting as a general p110 kinase inhibitor might produce an even better tumor-reduction effect when combined with EZH1/2 inhibitors.