Role of PI3K/AKT pathway in squamous cell carcinoma with an especial focus on head and neck cancers

PI3K/AKT pathway is an important pathway in the carcinogenesis since it has central impacts in the regulation of metabolic pathways, cell proliferation and survival, gene expression and protein synthesis. This pathway has been reported to be dysregulated in several types of cancers. In the current review, we summarize the role of this signaling pathway in squamous cell carcinomas (SCCs) originated from different parts of body cervix, oral cavity, head and neck and skin. The data presented in the current review shows the impact of dysregulation of PI3K/AKT pathway in survival of patients with SCC. Moreover, targeted therapies against this pathway have been found to be effective in reduction of tumor burden both in animal models and clinical settings. Finally, a number of molecules that regulate PI3K/AKT pathway can be used as diagnostic markers for different types of SCCs.


Introduction
PI3K/AKT pathway has important roles in the carcinogenesis since it has central impact in the regulation of metabolic pathways, cell proliferation and survival, gene expression and protein synthesis [1]. As a multimember family of heterodimeric lipid kinases, PI3Ks are classified into three distinct classes. Class IA PI3Ks are induced by receptor tyrosine kinases such as p110 catalytic subunit as well as p85-like regulatory subunits [1]. Class IB PI3Ks are induced by G protein-coupled receptors and regulatory subunits. Class II PI3Ks includes three proteins, namely PIK3C2A, PIK3C2B and PIK3C2G. Finally PIK3C3 is regarded as the single member of class III PI3Ks. PI3Ks can be induced by several upstream cell-surface receptors. In response to these stimuli, class I proteins catalyze the conversion of PI (4,5)P2 to the second messenger PIP3. AKT and PDK-1 serine/threonine kinases are two proteins that have PIP3-binding Pleckstrin homology (PH) domain and are associated with PI3K in a variety of cells [2,3]. AKT is an evolutionarily conserved serine protein kinase being attributed to the AGC subfamily. This protein has three structural domains, namely N-terminal PH domain, a short C-terminal tail comprising a regulatory hydrophobic motif (HM) and a linker section with a central kinase catalytic domain [6]. AKT family of proteins includes three homologous subtypes, namely AKT1-AKT3. In response to increase in PI (3,4,5)P3 levels and to a lesser extent accumulation of PI (3,4)P2, AKT is recruited on the cell membrane through its PH domain and exerts its catalytic roles through activation of a PDK1-induced threonine phosphorylation and mTORC2-mediated serine phosphorylation. These phosphorylation events occur at specific sites of AKT1, AKT2 and AKT3 [4,5]. The effects of AKT on regulation of important downstream effectors including FOXO, mTOR and GSK3b endows this molecule the ability to influence cell proliferation and survival, genome stability, and metabolic pathways [1]. PI3K/AKT pathway has been reported to be dysregulated in several types of cancers. In the current review, we summarize the role of this signaling pathway in squamous cell carcinomas (SCCs) originated from different parts of body cervix, oral cavity, head and neck and skin. Hou et al. have assessed the clinical outcomes of individuals with metastatic or recurrent cervical cancer during a phase I clinical trial. They have reported longer survival of patients with SCC of the cervix who had PIK3CA mutations compared with those without PIK3CA mutations. In fact, their results have shown that matched therapies against the activated PI3K/AKT/mTOR pathway have significant clinical benefit [6]. Another study in the context of cervical SCC has shown over-expression of the endogenous inhibitor of mTOR complexes DEPTOR in these cells and tissues. DEPTOR silencing has enhanced apoptosis of these cells via increasing expression of p38 MAPK and suppression of PI3K/AKT activity through feed-back suppression from mTORC1-S6K. Moreover, knock down of this gene has led to reduction of levels of nitric oxide synthases iNOS and eNOS, and enhancement of activity of ERK1/2 and p38 MAPKs. Moreover, DEPTOR could affect ERK1/2 expression in through modulation of AKT. Cumulatively, DEPTOR increases survival of cervical SCC cells and its knock down leads to cell apoptosis through distinctive impacts on PI3K/ AKT and p38 MAPK [7]. Moreover, the over-expressed receptor for advanced glycation endproducts (RAGE) has been shown to be involved in the pathogenesis of cervical SCC through modulation of PI3K/AKT activity. This protein has been found to promote proliferation of cervical SCC, enhance expression of PCNA, inhibit cell apoptosis along, reduce Bax/Bcl-2 ratio, and induce activity of PI3K/AKT pathway. RAGE silencing has reduced tumor burden in a xenograft model of cervical SCC. Finally, the PI3K inhibitor LY294002 could efficiently inhibit activity of PI3K and AKT, and suppress RAGE-induced pro-proliferative and anti-apoptosis effects [8]. Table 1 shows the role of PI3K/AKT pathway in squamous cell carcinoma of cervix.

Laryngeal squamous cell carcinoma (LSCC)
Mukonal, the isolated alkaloid from the plant Murraya koenigii has been shown to reduce the viability of laryngeal SCC cells, induce their apoptosis and arrest them at G2/M phase possibly through suppression of activity of PI3K/AKT and MEK/ERK pathways [20]. Moreover, dehydrocostus lactone isolated from Saussurea costus Lipech has been found to exert cytotoxic effects in this type of cancer. This substance could inhibit viability, migration and proliferation of laryngeal SCC cells without affecting viability of normal larynx epithelial cells. Notably, dehydrocostus lactone could promote function of p53 and P21 and induce cells apoptosis through suppression of PI3K/Akt/Bad pathway and stimulation of endoplasmic reticulum stress-mediated apoptotic pathways. In vivo assays have also verified these effects [21].
Another study has shown up-regulation of FGFR1, FGFR3 and PI3K/AKT kinase expression levels in the squamous cell laryngeal cancer samples compared with non-cancerous laryngeal mucosa specimens. Notably, over-expression of PI3K/AKT kinase has been associated with a high tumor front grading. Moreover, levels of the p-PI3K regulatory kinase protein have been associated with survival rate of patients. Taken together, FGFR1, FGFR3, and downstream regulatory kinases from the PI3K/AKT pathway might be regarded as putative markers indicative of invasive properties of laryngeal cancer [22]. Table 2 shows the role of PI3K/AKT pathway in laryngeal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC)
Expression analyses esophageal cancer tissues have shown up-regulation of miR-21, PI3K, and AKT, while down-regulation of PTEN in these tissues compared with adjacent non-cancerous tissues. Notably, samples obtained from patients with lymph node metastases and poor differentiation levels had lower expression of PTEN and higher levels of PI3K and AKT proteins. Suppression of miR-21 levels in esophageal cancer cells has led to up-regulation of PTEN, down-regulation of PI3K and AKT and reduction of proliferation rate, migration, and invasion of cells. This miRNA has been found to target PTEN. Cumulatively, miR-21 has been shown to target important molecules in PTEN/PI3K/AKT signal pathway, enhancing proliferation, migration, invasiveness, and cell cycle transition, and suppressing apoptotic pathways in esophageal SCC cells [47]. Another study in esophageal SCC patients has shown correlation between p-EGFR expression and all of the other phosphorylated biomarkers. Notably, gender, N stage, and expression levels of p-AKT1 have been independently correlated with overall survival of patients. In fact, over-expression of p-AKT1 has been found to be indicative of low survival. However, levels of EGFR and p-EGFR have not been correlated with patients' survival [48]. Moreover, dysregulation of PAFR via PI3K/AKT pathway has been reported to contribute to the progression of esophageal SCC [49].    On the other hand, vitamin E succinate could induce apoptosis of esophageal SCC cells through modulation of PI3K/AKT signaling this agent has decreased growth of EC109 cells by approximately 45 and 81% in concentrations of 10 and 100 µM, respectively [50]. Moreover, Dasatinib via suppressing the PI3K/AKT and STST3 pathways could improve sensitivity to cisplatin in esophageal SCC cells [51]. Table 3 shows the role of PI3K/AKT pathway in esophageal SCC. Figure 1 illustrates the aberrant expression of various miRNAs, which contribute to adversely modulating the PI3K/AKT signaling pathway involved in triggering several kinds of squamous cell carcinomas.

Pharyngeal squamous cell carcinoma (PSCC)
In patients with hypopharyngeal SCC, expression of p-Akt and p-Erk has been shown to be remarkably elevated parallel with progression of clinical stage, indicating the possible roils of PI3K/Akt and MAPK/ERK pathways in evolution and progression of this type of cancer. Notably, GDC-0980 and Refametinib have exerted cytotoxic effect on hypopharyngeal SCC cells. These agents could block cell cycle progression in G1 phase, reduce cyclin D1 and p-Rb levels and increase p27 levels. GDC-0980 could also inhibit migratory potential of these cells and reduce levels of p-PKCζ, p-Integrin β1 and uPA metastasis-related proteins. Taken together, dual suppression of PI3K/Akt and MAPK/ERK pathways by mentioned agents can be regarded as a possible strategy for treatment of hypopharyngeal SCC [60]. NVP-BEZ235 when combined with cisplatin could inhibit proliferation of hypopharyngeal SCC cells and arrest cell cycle at G2/M phase via modulation of the PI3K/AKT/mTOR pathway [61].
JARID1B, as a tumor suppressor, via the SHIP1/AKT pathway could improve differentiation of hypopharyngeal SCC cells and suppress their proliferation [62]. On the other hand, S100A11 could play an important role in the migration, carcinogenesis and protection of HPSCC from cell death induced by 5-Fu via the PI3K/AKT pathway [63]. Table 4 shows the role of PI3K/AKT pathway in pharyngeal squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC)/tongue squamous cell carcinoma (TSCC)
Lycopene has been revealed to inhibit proliferation, migration and invasiveness of oral SCC cells as well as in vivo growth of tumors. Moreover, this substance could suppress epithelial-mesenchymal transition and activate apoptotic pathways through decreasing activity of PI3K/AKT/mTOR signaling. These effects are exerted through enhancing expressions of E-cadherin and Bax and decreasing levels of N-cadherin, p-PI3K, p-AKT, p-m-TOR, and bcl-2 [69]. Thymoquinone has also been shown to suppress invasion, proliferation and migration of oral SCC cells and induce their apoptosis via inhibiting the PI3K/AKT pathway [70]. Moreover, Licochalcone A could suppress migration, invasion, and proliferation of oral SCC cells via modulation of the PI3K/AKT pathway [71].
A number of non-coding RNAs have been reported to exert their effects in the pathogenesis of oral SCC through modulation of this pathway. This speculation has been verified by knock-down experiments. For example, suppression of lncRNA MALAT1 could inhibit invasion, migration, and proliferation of TSCC cells via suppressing the PI3K/AKT pathway and down-regulating MMP-9 [72]. Moreover, circCDR1 has been shown to improve the viability of oral SCC cells by promoting autophagy via the AKT/ERK/mTOR pathway [73]. Table 5 shows the role of PI3K/AKT pathway in oral SCC.

Unidentified types of head and neck squamous cell carcinoma (HNSCC)
Expression of FKBP9P1 has been shown to be increased in HNSCC samples and cells. Over-expression of this gene has been correlated with advanced T, N and clinical stages as well as poor prognosis of affected individuals. FKBP9P1 silencing has suppressed proliferation, migratory potential, and invasiveness of these cells, possibly through inhibition of PI3K/AKT signaling [38]. PFN2 is another up-regulated gene in HNSCC and cells. PFN2 silencing has suppressed proliferation, invasiveness, and migratory potential of HNSCC cells, possibly through reduction of Akt and GSK-3β phosphorylation as well as decrease in β-catenin levels. In other words, PFN2 has been shown to promote proliferation and metastatic ability of HNSCC through inducing activity of the PI3K/Akt/ β-catenin pathway [39]. Similarly, DKK3 has been shown to increase the malignant properties of HNSCC via the PI3K/AKT/mTOR and MAPK pathways [40].
An in vitro study has shown that the anti-cancer agent osthole induces cell cycle arrest at G2/M phase and blocks proliferation of HNSCC cells via suppressing the PI3K/AKT pathway [41]. Finally, PI3K/AKT pathway has been shown to mediate the adaptive resistance to antiprogrammed death-1 (PD1) therapy through upregulating Tim-3 [42]. Table 6 shows the role of PI3K/AKT pathway in head and neck squamous cell carcinoma.

Cutaneous SCC
α-mangostin has been shown to suppress skin tumor formation and growth, decrease levels of pro-inflammatory molecules and increase levels of anti-inflammatory ones both in tumor and circulation. Notably, this substance could induce autophagy of skin cancer cells and regulate   [59] expression of autophagy-related proteins. Most notably, α-mangostin can inhibit activity of the PI3K/AKT/ mTOR signaling, as demonstrated by down-regulation of p-PI3K, p-Akt and p-mTOR [115]. Moreover, Lapatinib could suppress epithelial-mesenchymal transition in skin SCC via modulation of WNT/ERK/PI3K/AKT axis [116].
The anti-cancer effects of Lactucopicrin in skin cancer is also mediated through modulation of PI3K/AKT/mTOR pathway [103]. A number of non-coding RNAs can also modulate progression of skin SCC through influencing activity of PI3K/AKT pathway. For instance, miR-451a via PDPK1-mediated PI3K/AKT modulation could prevent progression of skin SCC [117]. Moreover, lncRNA LINC00520 via inactivating the PI3K/AKT pathway by downregulating EGFR could prevent the progression of this type of cancer [118]. Table 7 shows the role of PI3K/AKT pathway in skin SCC. Figure 2 represents the role of several ncRNAs in various types of SCCs via regulating the PI3K/AKT/mTOR signaling pathway. Mounting studies have revealed that dysregulation of PI3K/AKT signaling pathway can play a crucial role in the carcinogenesis especially in squamous cell carcinomas. A recent study has detected that overexpression of miR-433 could suppress the growth and metastasis of cervical cancer cells via down-regulating the FAK/PI3K/AKT signaling cascade, and could enhance the apoptosis and caspase-3/-9 function. Moreover, miR-433 could promote the expression levels of p53 and Bax, and inhibit that of MDM2 in cervical cancer [18]. Further experiment has validated that miR-132 plays an oncogenic role in laryngeal squamous cell carcinoma by suppressing the expression of FOXO1, p27, and p21. Overexpression of this miRNA could promote cell proliferation and tumor growth via up-regulating cyclin D1 as well as activating the PI3K/AKT pathway in LSCC cells [34]. Another research has pointed out that miR-21 could have a significant role in enhancing cell proliferation, migration, invasion, and cell cycle, and suppressing apoptosis of human esophageal cancer cells via down-regulating the expression of PTEN and activating PI3K/AKT signaling pathway [47]. Green lines indicate the positive regulatory effect among miRNAs and their targets, and red lines depict negative ones among them. All information regarding the role of these miRNAs involved in the modulation of PI3K/AKT signaling cascade in various types of squamous cell carcinomas can be seen in Tables 1-4

Discussion
PI3K/AKT has essential roles in the development of different types of SCC. Over-expression of PI3K, AKT, and p-mTOR has been reported in SCC tumors in association with down-regulation or absence of PTEN [122]. Gain of function mutations in constituents of this pathway, amplification of PIK3CA and AKT, overexpression of AKT and inactivating mutations or loss of PTEN are involved in the aberrant activity of this signaling pathway and subsequent progression of cancer {Simpson, 2015 #155}. Thus, identification of the underlying mechanism of over-activation of PI3K/AKT pathway in SCC has practical significance in design of novel therapeutic options. Moreover, a number of anti-cancer drugs such as cisplatin, LY294002, Licochalcone A, Mukonal, Dehydrocostus Lactone, Curcumin, Chloroquine, Osthole, Vitamin E succinate, Dasatinib, Tanshinone IIA, Genipin, Pristimerin, 5 fluorouracil, Sanguinarine, doxorubicin, AD198, Salvanic acid B, Oridonin, Plumbagin, Fig. 2 A schematic representation of the role of several ncRNAs in regulating the PI3K/AKT/mTOR signaling pathway in OSCC, TSCC and Cutaneous SCC. Accumulating evidence has revealed that various ncRNAs (lncRNAs, circRNAs, and miRNAs) could be negatively involved in triggering different kinds of SCCs via activating PI3K/AKT/mTOR signaling cascade. As an illustration, previous study has authenticated that up-regulation of lncRNA MALAT1 could promote the proliferation, migration, and invasion of tongue cancer cells via increasing the expression levels of AKT and MMP-9 [72]. Another finding confirms that overexpression of miR-21-5p could inhibit apoptosis via down-regulating the expression levels of PDCD4 as well as pro-apoptotic protein Bax and up-regulating FOXO1 and Bcl2 through directly activating the PI3K/AKT pathway in tongue squamous cell carcinoma [79]. Furthermore, mounting research has demonstrated that miRNA-451a via directly targeting PDPK1 could suppress cutaneous squamous cell carcinoma development by modulating the PI3K/AKT signaling pathway [117]. Green lines indicate the positive regulatory effect among ncRNAs and their targets, and red lines depict negative one among them. All the information regarding the role of these ncRNAs involved in the regulation of the PI3K/AKT signaling pathway in several kinds of squamous cell carcinomas can be seen in Tables 6, 7 a-mangostin, Lapatinib, Lactucopicrin and caffeic acid n-butyl ester have been found to exert their therapeutic effects in SCC via modulation of this pathway. It is worth mentioning that drug-loaded nanospheres and microspheres as a novel strategy for drug delivery can be used to form a material, mechanism, and cell combination that can not only treat the disease, but also verify the pathway. The possibility of using these systems for delivery of afore-mentioned drugs should be studies in future studies.
In brief, the bulk of evidence shows the impact of dysregulation of PI3K/AKT pathway in the pathogenesis of SCC and determination of survival of patients with this type of cancer. Moreover, targeted therapies against this pathway have been found to be effective in reduction of tumor burden both in animal models and clinical settings. Since this pathway has an established role in the induction of epithelial-mesenchymal transition, these therapies are expected to affect tumor metastasis as well. Besides, therapeutic modalities against PI3K/ AKT might act in a synergic manner with other anticancer modalities, enhancing the survival of affected individuals. PI3K/AKT pathway can also act as a mediator of HPV-induced cancer stem-like cells features in SCC [59], further highlighting the importance of this pathway in malignant features of SCC.
Finally, a number of molecules that regulate PI3K/ AKT pathway can be used as diagnostic markers for different types of SCCs.
Recent studies have also indicated the impact on noncoding RNAs in the regulation of PI3K/AKT pathway in different cancers, including SCC [123]. Thus, when designing novel therapeutic options against this pathway, it is necessary to consider the regulatory roles of these transcripts and their expression levels in these patients. Such approach may lead to establishment of a more effective personalized therapeutic strategy.