2.1. IP₆K1

IP₆K1 has been implicated in biological processes, such as energy metabolism, insulin signaling, trafficking, chromatin remodeling, cell migration, cancer metastasis, and neutrophil functions.

Recent studies suggest that in IP₆K1-KO mice models, IP₆K1 suppression increases energy expenditure by stimulating the protein kinase AMPK ^[37][38][27,78]. AMPK and Akt are significantly modulated under insulin stimulation ^[39][79]. IP₆K1 could modulate AMPK and Akt activities by interfering with insulin release. The link between IP₆K1 and Akt merits detailed discussion. Akt resides in the cytosol in an inactive conformation and translocases to the plasma membrane after cell stimulation. The Akt pleckestrin homology domain has a high affinity for PIP₃, which promotes Akt translocation to the membrane ^[40][80]. The Akt/PI3K interaction causes conformational changes and subsequent PDK1-dependent phosphorylation at the Thr³⁰⁸ kinase domain. However, full activation requires a further phosphorylation at S473, catalyzed by several enzymes, including PDK2 and ILK. IP₇ competitively binds to the PH domain, thus preventing its phosphorylation and activation by PDK1. Notably, IP₇ strongly inhibits Akt activation, with an IC50 of 20 nM, close to the Kd (35 nM) displayed by PIP₃ in respect to the PH domain of Akt ^[41][81]. IP₆K1 knockout leads to increased PDK1-dependent Akt activation, determining a plethora of biochemical consequences for metabolic regulation, not yet well investigated. Indeed, after glucose stimulation and subsequent increase in the ATP/ADP ratio, a significant increase in IP₇ was observed. In detail, IP₇ production by IP₆K1 inhibits the stimulatory effect of IP₆ on AMPK. The response of IP₇ to the increase in ATP/ADP ratio occurs a few minutes (10–30) after the stimulus. In turn, IP₇ associates with the Akt PH domain, preventing interaction with PIP₃ and therefore reducing Akt membrane translocation and consequent insulin-stimulated glucose uptake. This mechanism involves feedback, whereby increased availability of ATP drives the system to inhibit glucose uptake by modulating insulin transduction by blocking Akt membrane recruitment ^[42][43][44][82,83,84]. This regulation may also be indirectly affected by IP₇-promoted nuclear localization of LKB1. Nuclear transfer of LKB reduces LKB cytosolic activity, thus hindering AMPK phosphorylation and activation ^[45][85]. It is worth noting that RNAi silencing of IP₆K1 blocks IP₇ and insulin release after glucose stimulation. In IP₆K1-KO models, changes in the intracellular IP₆/IP₇ ratio increase AMPK activation ^[46][86]. Conversely, Akt signaling is significantly increased, leading to a decrease in GSK3b phosphorylation, and augmented protein translation. Reduction in GSK3b phosphorylation increases its catalytic activity and is likely be followed by a surge in adipogenesis and diminished glycogen levels ^[47][87]. Indeed, after insulin stimulation, IP₇ decreases (from 33% to 60%) in IP₆K1 knockout hepatocytes, whereas Akt and GSK3β increase, improving glucose tolerance, presumably due to a decrease in hepatic glucose production ^[48][26]. Conversely, overexpression of IP₆K1 finally impairs insulin-signaling transduction, whereas IP₆K1 silencing may lead to insulin hypersensitivity, as observed in IP₆K1 KO mice. As proof of concept, a number of animal models of insulin hypersensitivity share the common biochemical signature of an increased tier of Akt activation and translocation ^[49][88]. Furthermore, in mouse embryo fibroblasts (MEFs), IP₆K1-induced energy expenditure inhibition leads to reduction of glycolysis via IP₇-mediated destabilization of the interaction between the transcriptional activators of glycolytic genes (GCR1 and GCR2) ^[50][25].

Although IP₆K2 proves sensitive to ATP/ADP fluctuations and may induce IP₇ synthesis, it is unlikely that it could act as a sensor of energy requirements, as does IP₆K. This apparent conundrum can be explained if we consider the cell compartmentalization of IP₆K. In fact, while IP₆K1 is usually found in the cytosol and nucleus, IP₆K2 is almost all in the nucleus ^[51][89].

2.2. IP

₆

K2

A number of studies suggest an essential role for IP₆K2 in cell death, migration, cancer metastasis, and progression. IP₆K2 activity sensitizes a number of cancer cells, including OVCAR3, HeLa, HEK293, PC12, and HL60, to apoptosis ^{[52][53][54][55]}[121,122,123,124]. Deletion of IP₆K2 prevents apoptotic consequences of γ-irradiation or β-interferon addition to ovarian cancer cells, while overexpression of IP₆K2 significantly raises cell death rate under the same conditions ^[53][122]. Overexpression of IP₆K2 augments the cytotoxic effects of many cell stressors, whereas transfection with a dominant negative IP₆K2 decreases cell death. It is noteworthy that the apoptosis surge is associated with increased synthesis of IP₇ and transfer of IP₆K2 from nuclei to mitochondria, while no changes are recorded in the intracellular localization of the other IP₆K isoforms ^[52][121]. In detail, IP₆K2 directly mediates IFNβ-induced apoptosis ^[52] [121] by enzymically regulating p53 activity and by increasing expression of the Apo2L/TRAIL ligand that initiates apoptosis through death-receptor signaling. Namely, HSP90 physiologically binds IP₆K2 and inhibits its catalytic activity. By interfering with HSP-IP₆K2 binding, HSP90 fosters IP₆K2 activation that ultimately leads to increased cell apoptosis ^[56][21]. Nuclear localization of IP₆K2, promoted by interaction with HSP90, is a mandatory step for establishing proper IP₆K2-p53 binding. ^[57][22]. Indeed, IP₆K2 has been demonstrated to directly modulate p53-dependent apoptosis. Gene disruption of IP₆K2 in colorectal cancer cells selectively impairs p53-mediated cell death and favors cell cycle arrest ^[57][22]. This interaction suppresses phosphorylation of the cell cycle arrest regulator (p21) and its transcription, while enhancing p53-mediated apoptosis ^[58][23]. This implies that IP₆K2 acts as a switching factor, driving p53 activity towards apoptosis rather than cell cycle arrest. It should be noted that although IP₆K2 regulates p53 by direct binding, its catalytic activity generating IP₇ is essential for its influence on p53 signaling. It has also been observed that IP₆K2 can promote apoptosis independently of its enzyme activity. By interacting with TRAF2, IP₆K2 interferes with apoptosis and nuclear factor kappa β (NF-kβ) signaling, thus affecting the release of tumor necrosis factor α (TNFα) ^[27][33]. The proapoptotic activity of IP₆K2 is successfully antagonized by heat-shock proteins (HSPs). Overall, these findings suggest that IP₆K2 actively participates in the regulation of the Apo2L/TRAIL cell death pathway. Moreover, PP-IPs modulate cell death and telomere length in yeast by antagonizing the homolog of ataxia telangiectasia mutated (ATM) kinase, a regulator of the DNA damage response and apoptosis in mammals ^[59][125].

As strong as IP₆K2-mediated apoptosis may be, IP₆K2 participation in the regulation of such functions through its nuclear ^[60][126], mitochondrial ^[53][122], and cytosolic ^[54][61] [123,127] localization requires further investigation.

As observed in IP₆K1-KO models, IP₆K2-KO, too, reduces cell–cell adhesion, growth, spreading, metastasis, and FAK phosphorylation in cancer cells. The molecular mechanisms so far proposed include LKB1 sequestering in the nucleus and inhibition of cytosolic phosphatase activation, and consequently, FAK dephosphorylation ^[59][125]. Remarkably, IP₆K1 and IP₆K2 both favor sequestering of LKB into the nucleus in an inactive form ^[45][61][85,127].

The tumor suppressor LKB1 is credited with inhibiting FAK activation ^[62][128] and enhancing E-cadherin expression ^[63][129], thus inhibiting motility and invasiveness. These findings strongly suggest that LKB1 plays a critical role in controlling the balance between cell–cell and cell–matrix adhesion. In addition, by modulating AMPK activity, LKB1 interferes with a number of critical metabolic processes ^[64][130]. Interaction with two subunits of the heterotrimeric holoenzyme (STRAD and Mo25) in the cytosol leads to phosphorylation of LKB1 at serine-428 and then activation by PKCδ ^[65][131]. This finding is worth mentioning as it suggests that IP₆K2/IP₇ can fine tune the activity of “constitutive” kinases, like PKCδ and CK2 ^[37][27], as previously indicated.

Indeed, a number of results have clearly established that deletion of IP₆K1 or IP₆K2 reduces cell migration, while IP₆K2-KO, quite paradoxically, reduces tumor volume ^[66][133]. IP₆K2-KO cells display almost total loss of IP₈ levels, whereas only a small decrease in IP₈ levels was recorded in IP₆K1-KO ^[58][67][23,113]. It is tempting to speculate that persistent IP₇ synthesis, even at a lower rate, is mandatory for apoptosis, as previously suggested. However, somewhat paradoxically, complete suppression of IP₆K2 enhances development of carcinoma of the gastrointestinal tract in mice ^[68][134], probably because IP₆K2-dependent pyrophosphate synthesis may in turn activate p53 and protein kinase CK2, thus promoting apoptosis [2]. In IP₆K2 knockout mice, a substantial increase in tumorigenesis in response to 4-nitroquinoline-1-oxide, a UV-mimetic carcinogen, has been observed ^[69][135]. These findings provide indirect confirmation of the link between IP₆K2 and p53, as p53-mediated apoptosis is required for apoptosis induced by UV-mimetic factors. However, unlike p53 knockouts models, the IP₆K2 mutants do not develop spontaneous tumors. This apparently odd behavior suggests that IP₆K2 may only influence p53 proapoptotic activity when the system is exposed to a carcinogen stressor but does not directly entail “spontaneous” carcinogenesis.

In ovarian carcinoma cells, IP₆K2 deletion confers protection against interferon alpha (IFNα)-induced cell death, whereas overexpression of IP₆K2 enhances the apoptosis rate promoted by IFNα and/or γ-irradiation ^[70][136]. Yet some controversial results have also been reported, since under estradiol stimulation, β-catenin-induced oncogenesis significantly increases IP₆K2 gene expression downstream of the Wnt/β-catenin signaling pathway ^[71][137]. Overexpression of IP₆K2 presumably leads to increased pyrophosphate synthesis, reducing cell levels of IP₆, which may in turn contribute to the transformed phenotype. On the other hand, suppression of IP₆K1 confers protection against tumors experimentally induced with carcinogens ^[72][138].

Although these findings are still preliminary, they suggest that IP₆K1 and IP₆K2 can exert opposite effects in carcinogenesis. It is also likely that the effects of IP₆K2 on cancer cells are disjointed, i.e., IP₆K2 probably enhances apoptosis while increasing the acquisition of an invading/migrating phenotype. IP₆K2 may, therefore, act as a tumor suppressor in the initiation stage but contribute to metastatic spread by enacting EMT at later stages. It is worth underlining that similar dual roles have been observed for TGF-β1 ^[73][139].

2.3. IP

₆

K3

IP₆K3 is highly expressed in mouse and human myotubes and muscle ^[74][77]. Its physiological role is relatively unexplored. High levels of expression have been detected in the brain. Purkinje cells regulate motor learning and coordination, and IP₆K3 deletion alters these functions. Abnormalities in cell size and spine density are detected, perturbed by dysfunctional IP₆K3 binding of adducin and spectrin, two cytoskeletal proteins involved in the morphogenesis of dendritic trees ^[74][77]. Regarding other IP₆Ks, IP₆K3 seems to participate somehow in glucose metabolism. Indeed, IP₆K3-null mice exhibit lower blood glucose and reduced insulin levels, associated with increased plasma lactate levels. These findings suggest that downregulation or suppression of IP₆K3 can enhance glycolysis. However, IP₆K3 suppression is followed by a significant reduction in pyruvate dehydrogenase kinase-4 (PDK4) ^[75][140]. Since PDK4 depresses glucose oxidation by inhibiting conversion of pyruvate to acetyl coenzyme A (acetyl-CoA), it is paradoxical that IP₆K3 suppression does not lead to an increase in glucose oxidation.

3. Future Perspectives

Growing interest focused on IPs has shed light on their biological functions and corresponding deregulation issues. Among IPs, IP₇ plays a significant role in cell metabolic balance, ATP production, and phosphate homeostasis. From these studies, IP₆Ks emerge as key regulators of IP₇ intracellular levels in physiological and pathological processes (Figure 2).

Figure 2. IP₆Ks and their pathways. IP₃ is metabolized in many inositol polyphosphates, of which IP₆ is the most abundant. IP₆Ks produce IPs (IP₇) starting from IP₆. IP₆K/IP₇ levels are crucial for regulating various biological processes. IP₆K1 binds α-actinin localized at focal adhesions, promoting its phosphorylation by FAK and regulating cell migration. IP₆-stimulated AMPK activation is inhibited by high levels of IP₇, reducing cytosolic localization of LKB. High PA levels promote nuclear IP₆K1 translocation, inhibiting ISYNA1, and consequently, de novo biosynthesis of myo-inositol. Nuclear IP₆K1 interacts with JMJD2C and induces its dissociation from chromatin, increasing H3K9me3 levels and inhibiting transcription of target genes. Likewise, IP₆K2 may localize in the nucleus, downstream of its interaction with HSP90. In turn, nuclear IP₆K2 localization promotes binding to p53, suppressing p21 activation and transcription.

Interest in the development of molecular factors that can (selectively) interrogate and manipulate the cell actions of inositol pyrophosphates, especially by modulating IP₆Ks and PPIP₅Ks, is gaining momentum ^[70][136]. Targeting these pathways could be helpful in certain diseases but also potentially dangerous. For example, knockout experiments on IP₆Ks highlighted a worse situation in mice, sensitizing the animals to chemical tumorigenesis ^[69][135], lung inflammation ^[76][112], and loss of motor learning, coordination, and fitness ^[74][77][77,141]. It is therefore crucial to determine whether pharmacological inhibition of IP₆Ks is safe enough to pursue clinical investigations.

Studies based on gene deletion assays are unlikely to provide useful data, since more than 900 genes are altered by deletion of IP₆K homolog (Kcs1) in S. cerevisiae ^[78][54]. The range of this genetic penetration probably highlights the functional polyvalence of IP₆Ks, which presumably have both catalytic and scaffolding functions, as already demonstrated for inositol pentakisphosphate kinase ^[79][142] and inositol polyphosphate multikinase ^[80][143]. A more promising approach may focus on specific cell-permeant inhibitors of PP-IPs or on “physiological” modulators of IP₆Ks, an approach that at least in principle would not be flawed by secondary genetic changes or interference with IP₆K scaffolding functions.

The compound N2-(m-trifluorobenzyl)N6-(p-nitrobenzyl)purine (TNP) has been shown to bind specifically to IP₆Ks by competing with ATP for the same binding site. As a result, TNP reduces IP₇ levels by inhibiting the kinase and phosphatase activities of IP₆Ks. Within 2 h of treating various cell types with 10–30 μM TNP, levels of IP₇ fell by 60–90% ^[81][82][67,144], and IP₈ synthesis was also significantly reduced ^[83][145]. As expected, IP₆ levels increased proportionally by as much as 40%.

TNP does not efficiently cross the blood–brain and blood–testis barriers. In fact, chronic TNP administration (15 weeks, 10 mg/kg/day) in mice does not lead to neuronal or reproductive abnormalities ^[84][146]. However, TNP could interfere with the metabolism of other drugs by inducing modifications in drug signaling or increasing Ca²⁺ and Zn²⁺ levels ^[85][147].

TNP inhibitory activity discriminates between IP₆Ks and other inositol phosphate kinases (IPMKs and IP₃Ks). The catalytic site of the IP₆K family is structurally related to that of IPMKs and IP₃Ks, though IP₆Ks have around 100-fold lower affinity for ATP than do the latter ^[82][144]. Higher TNP values are therefore required to efficiently neutralize IP₃K (IC50 0.47 μM for IP₆Ks versus 18 μM for IP₃K). However, TNP displays some off-target effects, including ERK phosphorylation, which in principle is not mediated by IP₆Ks. The use of TNP to investigate the intracellular functions of IP₆Ks is therefore debatable. To minimize undesirable effects, it could be useful to develop safe and selective inhibitors of IP₆K isoforms for investigating the specific role sustained by the different IP₆K isoforms.

Regarding carcinogenesis, IP₆K1 and IP₆K2 activities presumably drive cells and tissues towards opposite outcomes. As previously reported, IP₆K1 joins in Akt signaling, and its knockout decreases IP₇ synthesis, resulting in enhanced PDK-dependent phosphorylation of Akt activation. Hyperactivation of Akt (∼10- to 50-fold) ^[86][87][148,149] is known to enable tumorigenesis ^[88][150]. However, IP₆K1-KO is only associated with a minimal increase in Akt activation in mice ^[37][27], insufficient to enact neoplastic development ^[37][27]. Indeed, it has been reported that deletion of IP₆K1 protects against chemical tumorigenesis and metastasis ^[67][113], although the mechanisms underlying the effect are still unknown. Instead, IP₆K2-KO sensitizes to chemical tumorigenesis and probably increases the occurrence of spontaneous cancer ^[72][138].

 

 

Acronyms: 1-IP₇ (1-diphospho-2,3,4,5,6-pentakisphosphate); 5-IP₇ (5-diphospho-1,2,3,4,6-pentakisphosphate); Akt (protein kinase B); AMPK (5’ AMP-activated protein kinase); DAG (diacylglycerol); FAK (focal adhesion kinase); H3K9me3 (histone 3 lysine 9 trimethylation); IP₂ (inositol-2-phosphate); IP₃ (inositol-3-phosphate); IP₄ (inositol-4-phosphate); IP₅ (inositol-5-phosphate); IP₆ (inositol-hexakisphosphate or phytic acid); IP₆K1 and IP₆K2 (inositol hexakisphosphate kinase 1/2); IPK1 (inositol-pentakisphosphate 2-kinase); IPMK (inositol polyphosphate multikinase); ISYNA1 (d-3-myoinositol-phosphate synthase); JMJD2C (Jumonji domain-containing protein 2C); LKB (liver kinase B1); P (phosphate group); PA (phosphatidic acid); PI3K (phosphatidylinositol 3-kinase); PIP₂ (phosphatidyl-inositol-4,5-biphosphate); PIP₃ (phosphatidylinositol-3-phosphate); PKC (protein kinase C); PLC (phospholipase C); PPIP₅K (inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinase); PTEN (phosphatase and tensin homolog); P₈ (1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate); G6P (glucose-6-phosphate); IP₁ (inositol-1-phosphate, myo-Inositol); ITPK1 (inositol-tetrakisphosphate 1 kinase).