The Role of the Dysfunctional Akt-Related Pathway in Cancer: Establishment and Maintenance of a Malignant Cell Phenotype, Resistance to Therapy, and Future Strategies for Drug Development

Akt serine/threonine kinases, or PKB, are key players in the regulation of a wide variety of cellular activities, such as growth, proliferation, protection from apoptotic injuries, control of DNA damage responses and genome stability, metabolism, migration, and angiogenesis. The Akt-related pathway responds to the stimulation mediated by growth factors, cytokines, hormones, and several nutrients. Akt is present in three isoforms: Akt1, Akt2, and Akt3, which may be alternatively named PKBα, PKBβ, and PKBγ, respectively. The Akt isoforms are encoded on three diverse chromosomes and their biological functions are predominantly distinct. Deregulations in the Akt-related pathway were observed in many human maladies, including cancer, cardiopathies, neurological diseases, and type-2 diabetes. This review discusses the significance of the abnormal activities of the Akt axis in promoting and sustaining malignancies, along with the development of tumor cell populations that exhibit enhanced resistance to chemo- and/or radiotherapy. This occurrence may be responsible for the relapse of the disease, which is unfortunately very often related to fatal consequences in patients.


Introduction
Akt serine/threonine protein kinases are also termed PKB and constitute fundamental intracellular signaling systems for the regulation of an ample assortment of cellular and physiological activities, such as cell growth, proliferation, protection from apoptosis, modulation of DNA damage response and genome stability, motility, angiogenesis, and metabolism [1][2][3][4][5][6][7]. These Akt-mediated cellular functions are regulated by various types of external stimuli, which derive from the interaction of growth factors, hormones, cytokines, and nutrients with specific cellular receptors [1][2][3][4][5][6][7]. Some of the main hormones and growth factors that have the ability to stimulate the Akt axis comprise epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and nerve growth factor (NGF) [1][2][3][4][5][6]. Basically, the interaction between the external factors and the Akt axis occurs via ligand-cellular receptor binding, which, in turn, results in the transient Akt phosphorylation, with consequent temporary activation of the Akt intracellular signaling system. Overall, the Akt stimulation mediated by growth factors regulates cell cycle transition from G1/S to G2/M phase [1][2][3][4][5][6][7]. In addition, the Akt-related pathway comes into play in the orchestration of the DNA damage response and cellular genome stability [7]. Intracellular upstream effectors that activate the Akt-related pathway include phosphatidylinositol 3-kinase (PI3K) [8][9][10], LKB1 [11], and phosphatase and tensin homologue deleted on chromosome ten (PTEN) [12], whereas downstream regulators consist of mammalian target of rapamycin (mTOR) [13][14][15], eukaryotic initiation factor 4E (eIF4E) [16,17], and tuberous sclerosis complex 2 (TSC2) [18][19][20][21]. Genetic analysis revealed that cellular protooncogenes encode Akt, eIF4E and the PI3K p85a regulatory subunit and p110 catalytic domain [1-10, 16, 17]. Instead, tumor suppressor genes encode TSC2, PTEN, and LKB1 [11,12,[18][19][20][21]. In recent years, a variety of studies conducted in Akt isoform-specific knockout mice unequivocally demonstrated that the biological functions of the three Akt isoforms are for the most part dissimilar from one another [2,[27][28][29][30][31]. For example, Akt1 is essential for cell survival, as Akt1-null cells are more susceptible to apoptosis than Akt1-positive cells and Akt1 knockout mice are substantially smaller than wild-type littermates [32,33]. Instead, Akt2 has a more prevalent role in the regulation of glucose homeostasis, as Akt2 knockout mice exhibit higher incidence of a type-2 diabetes-like illness and primary cell cultures derived from these animals show evident ineffective glucose consumption [34,35]. Akt3 has a more predominant purpose in postnatal brain development, as Akt3 knockout mice exhibit a median 25% reduction in brain weight and size, even though no major anatomical deformities were reported in this study, besides a considerable decrease of white matter fiber connections in the corpus callosum [36]. Another report demonstrated that Akt2 has the ability to enhance the resistance of rod photoreceptor cells to apoptotic injuries that may be caused by light-related stress, whereas the other two Akt isoforms lack this property [28]. These findings were observed in knockout mice models, which also showed that light-induced cell stress specifically activates Akt2 [28]. Intriguingly, Akt1 is essential to enhance cell survival for the majority of cells, except for lightinduced cell stress in rod photoreceptor cells, which explicitly necessitate the activation of Akt2.
On these grounds, the three Akt isoforms exhibit differential biological characteristics and kinase activities, which are in function of the cellular context. In addition, a defective and less active Akt-related pathway does not provide an efficient protection from apoptotic injuries, which may become a contributing factor in the pathogenesis and/or clinical progression of several human maladies, such as neurodegenerative diseases [37][38][39][40][41], illnesses of the cardiovascular system [42][43][44][45], and type-2 diabetes [33,34,46]. Conversely, the overexpression and/or constitutive enhanced activity of the Aktrelated pathway were observed in a wide variety of human tumors [1,2,22,23,30,[47][48][49][50][51][52][53][54][55]. This paper discusses the implications of deregulations in the Akt signaling system that were reported in different types of cancer.
The protooncogene TCL1 boosts the stimulation of the Akt axis activity through binding to the Akt PH domain [22]. Under normal physiological conditions, TCL1 expression is confined to cell populations of the immune system, during the early stages of development [22]. The increment of TCL1 expression levels in somatic cells is correlated with aberrant Akt kinase activity, as reported in different types of hematological malignancies and seminoma [22,55]. Moreover, TCL1 mediates Akt nuclear translocation [81]. The biological functions of nuclear Akt are currently under investigation [81,82]. It has been proposed that the presence of Akt in the nucleus is instrumental in inhibiting apoptosis, by blocking the caspaseactivated deoxyribonuclease [83].
An early study showed that Akt2 overexpression transformed mouse fibroblast NIH/3T3 cells [84], whereas another report indicated that Akt2 overexpression increased substantially metastatic features and invasion both in human breast cancer and human ovarian cell lines [85]. Conversely, Akt1 and Akt3 overexpression failed to reproduce the effects that were observed for Akt2 overexpression in the previously mentioned human tumor cell lines [85]. This is a further evidence that accounts for the nonredundancy of the three Akt isoforms.
Some studies showed an involvement of aberrant PI3K/Akt3 activity in human melanoma [50,86]. For instance, 70% of biopsies derived from patients with melanoma exhibited abnormal activities in the PI3K/Akt3-related signaling system [50]. A subsequent report showed that an enhanced PI3K/Akt3 pathway activity is one of the main contributors in the genesis of melanoma [86]. Moreover, several other studies supported the implication of the deregulated PI3K/Akt pathway in the development and/or clinical progression of melanoma [87][88][89][90][91].
In addition to enhanced levels of Akt expression, a number of Akt activating mutations were reported in various types of human cancers. For instance, a transforming point mutation that changes a single glutamic acid to lysine at amino acid residue 17 (E17K) within the PH domain confers a continuous state of activation in Akt1 [113]. This somatic point mutation was identified in human breast, ovarian, and colorectal tumors [113]. Intriguingly, the E17K point mutation was absent in Akt2 and Akt3 in the previously mentioned tumors [113], although an analogous point mutation in the Akt3 PH domain was found in human melanoma [114].
A deregulated Akt activity is among the main factors that are implicated in the establishment of a malignant phenotype and/or progression of the clinical course of the disease [1-6, 22, 23, 47-53]. On these grounds, the Akt-related pathway may be considered a suitable target for cancer therapy [52,55,57,79,[124][125][126]. However, the inhibition of the Akt axis is one of the requirements for enhanced cell motility [127][128][129][130][131][132]. In fact, the Akt signaling system suppresses the activity of the nuclear factor of activated T cells (NFAT) [127][128][129][130][131], which is a transcription factor that increases both cell motility and invasion in different kinds of malignancies [127][128][129][130][131][132][133][134][135]. Most likely, the Akt-induced inhibition of NFAT activity occurs through the Akt-mediated stimulation of the E3 ubiquitin-protein ligase Mdm2, which, in turn, promotes the degradation of NFAT [132]. Thus, the pharmacological inhibition of the Akt-related pathway in cancer therapy might unexpectedly become a contributing factor for the dissemination of cancer metastases [132]. Indeed, this is a very important aspect that should be taken under consideration in the planning of various therapeutic strategies for the treatment of malignancies in patients.
In recent years, a number of PI3K/Akt/mTOR inhibitors have been developed for the treatment of different types of tumors [199][200][201]. Some PI3K/Akt/mTOR inhibitors comprise rapamycin, sirolimus, metformin, everolimus, and temsirolimus [199][200][201]. Although the PI3K/Akt/mTOR axis is a promising target for the treatment of cancer, randomized phase III clinical trials reported suboptimal beneficial therapeutic effects in patients [199][200][201]. Exceedingly high levels of toxicity were unfortunately observed in various clinical trials [199][200][201]. Moreover, the effects of the inhibition of the PI3K/Akt/mTOR pathway can be circumvented in cancer cells through the Raf/MEK/ERK signaling system [200], which may protect malignant cells from drug-induced proapoptotic injuries and, therefore, produce chemoresistant cancer cells variants [200].
A study has recently shown that flavopiridol triggered a considerable Akt-Ser473 phosphorylation in human glioblastoma T98G cell line [194]. In contrast, as expected, flavopiridol treatment caused a reduction of Akt-Ser473 phosphorylation in human glioblastoma U87MG cell line and in human prostate cancer PC3 cell line [194]. As already discussed, Akt-Ser473 phosphorylation is a characteristic of the Aktrelated pathway activation, which, in turn, may protect cells from apoptotic injuries [1][2][3][4][5]. Flavopiridol is a pan-inhibitor of cyclin-dependent kinases and has been used in several clinical trials for the treatment of patients with various kinds of malignancies, albeit with modest therapeutic efficacy [57,[227][228][229][230]. The use of flavopiridol is supposed to impair the cellular signaling systems for protection from apoptosis and survival [57,[227][228][229][230]. However, the previously mentioned study on human glioblastoma T98G cell line indicates that flavopiridol might paradoxically play a relevant role in the production of tumor cell variants with enhanced resistance to chemotherapy, through increased activation of the Aktrelated pathway [57,194]. For this reason, various anticancer drugs should be screened to assess whether or not they may incidentally induce the increment of Akt-Ser473 phosphorylation in different types of human tumor cells [194].
Interestingly, it was also reported that a deregulated Akt axis has the ability to confer radioresistance to malignant cells by orchestrating DNA repair through nonhomologous end joining (NHH) [7]. In this regard, a group of investigators observed a substantial -radiation-induced increment of Akt-Ser473 phosphorylation in a variety of human glioblastoma cell lines, such as U87MG, MO59J, and LN-18 [231].
Investigations are currently underway to determine the mechanisms of flavopiridol and/or -radiation-induced enhancement of Akt-Ser473 phosphorylation in human glioblastoma cell lines. In fact, a better understanding of these mechanisms may lead to the identification of novel therapeutic targets, which can be eventually suppressed with new drug formulations, in order to prevent the constitution of cancer cell variants that are more resilient to chemo-and/or radiotherapy.

Conclusion
Undeniably, a deregulated Akt pathway is an important factor in the establishment and/or maintenance of a malignant cell phenotype. Moreover, a constitutively activated Akt axis is involved in the generation of tumor cell variants with enhanced resistance to chemotherapeutic agents and/or radiotherapy.
On one hand, an abnormal Akt-related pathway is a very promising target to implement therapeutic approaches for the treatment of different types of cancer. On the other hand, the repression of the deregulated Akt signaling system, per se, does not seem to be sufficient for an effective therapy and may pose a number of collateral issues. For instance, a drug-induced inhibition of the Akt activity in malignant cells may unexpectedly contribute to the formation and/or dissemination of cancer metastases [127][128][129][130][131][132]. Another quite unforeseen side effect of the Akt pharmacological targeting is related to the flavopiridol-induced increment of Akt-Ser473 phosphorylation in human T98G glioblastoma cell line [194]. In addition, an increased Akt-Ser473 phosphorylation was observed following -irradiation of a panel of human glioblastoma cell lines [231]. All of these findings, taken together, suggest the pursuit of combinational therapeutic approaches for the treatment of different types of cancer [232][233][234][235][236][237][238], in order to prevent as much as possible treatmentrelated side effects that may paradoxically contribute to the spreading of metastases and/or to the generation of cancer cell variants with higher resistance to therapeutic interventions.

Conflict of Interests
The author declares that there is no conflict of interests regarding the publication of this paper.