Mutational analysis of PTEN/PIK3CA/AKT pathway in oral squamous cell carcinoma

The phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma (AKT) viral oncogene pathway is involved in regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes associated with the PI3K/AKT pathway including PI3K, AKT, RAS and PTEN, are infrequently found within head and neck squamous cell carcinoma and more specifically are rarely reported in oral squamous cell carcinoma (OSCC) cases. We aimed to investigate the frequency of mutations in AKT1, PTEN, PIK3CA, and RAS (K-RAS, N-RAS, H-RAS) genes in 37 cases of oral squamous cell carcinoma (OSCC). Mutational analysis of PTEN, RAS, PIK3CA and AKT genes was performed using chip-based matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and by direct sequencing. The only gene mutated in our series was the PIK3CA. Missense mutations of the PIK3CA gene were found in 4 of our cases (10.8%); no correlation has been found with oral location, stage and survival. The absence of mutations in AKT1, PTEN, and RAS genes in the present study is in accordance with previous studies confirming that these genes are rarely mutated in OSCC. Our data confirm that PIK3CA is important to OSCC tumorigenesis and can contribute to oncogene activation of the PIK3CA/AKT pathway in OSCC. The knowledge of the PIK3CA's involvement in OSCC is important because a specific kinase inhibitor could be considered as a future therapeutic option for OSCC patients with PIK3CA mutations.     

FAM83B-mediated activation of PI3K/AKT and MAPK signaling cooperates to promote epithelial cell transformation and resistance to targeted therapies

Therapies targeting MAPK and AKT/mTOR signaling are currently being evaluated in clinical trials for several tumor types. However, recent studies suggest that these therapies may be limited due to acquired cancer cell resistance and a small therapeutic index between normal and cancer cells. The identification of novel proteins that are involved in MAPK or AKT/mTOR signaling and differentially expressed between normal and cancer cells will provide mechanistically distinct therapeutic targets with the potential to inhibit these key cancer-associated pathways. We recently identified FAM83B as a novel, previously uncharacterized oncogene capable of hyperactivating MAPK and mTOR signaling and driving the tumorigenicity of immortalized human mammary epithelial cells (HMEC). We show here that elevated FAM83B expression also activates the PI3K/AKT signaling pathway and confers a decreased sensitivity to PI3K, AKT, and mTOR inhibitors. FAM83B co-precipitated with the p85α and p110α subunits of PI3K, as well as AKT, and increased p110α and AKT membrane localization, consistent with elevated PI3K/AKT signaling. In tumor-derived cells harboring elevated FAM83B expression, ablation of FAM83B decreased p110α and AKT membrane localization, suppressed AKT phosphorylation, and diminished proliferation, AIG, and tumorigenicity in vivo. We propose that the level of FAM83B expression may be an important factor to consider when combined therapies targeting MAPK and AKT/mTOR signaling are used. Moreover, the identification of FAM83B as a novel oncogene and its integral involvement in activating PI3K/AKT and MAPK provides a foundation for future therapies aimed at targeting FAM83B in order to suppress the growth of PI3K/AKT- and MAPK-driven cancers.     

Epithelial V‐like antigen 1 promotes hepatocellular carcinoma growth and metastasis via the ERBB‐PI3K‐AKT pathway

The role of epithelial V‐like antigen 1 (EVA1) has been well studied in thymic development and homostasis; however, its putative relationship with cancer remains largely unknown. Therefore, here we investigated the role of EVA1 in hepatocellular carcinoma. Interestingly, EVA1 expression was significantly increased in hepatocellular carcinoma (HCC) and was also associated with a poor prognosis and recurrence in HCC patients. Overexpression of EVA1 promoted cell growth, invasion and migration in vitro. Consistently, knockdown of EVA1 expression inhibited proliferation and migration in vitro, while repressing metastasis of HCC cells in vivo. RNA‐seq analysis indicated that EVA1 is able to upregulate the expression of genes in the ERBB3‐PI3K pathway. Accordingly, an increased level of AKT phosphorylation was detected in HCC cells after EVA1 overexpression. LY294002, a PI3K inhibitor, inhibited AKT phosphorylation and rescued the tumor‐promoting effect of EVA1 overexpression. Altogether, the present study has revealed the oncogenic role of EVA1 during HCC progression and metastasis through the ERBB‐PI3K‐AKT signaling pathway, reiterating the potential use of EVA1 as a therapeutic target and/or prognostic marker for HCC.     

Gene alterations in the PI3K/PTEN/AKT pathway as a mechanism of drug-resistance (review)

The most common therapeutic approach for many cancers is chemotherapy. However, many patients relapse after treatment due to the development of chemoresistance. Recently, targeted therapies represent novel approaches to destroy cancer cells. The PI3K/PTEN/AKT pathway is a key signaling pathway involved in the regulation of cell growth. Dysregulated signaling of this pathway may be associated with activating mutations of PI3K-related genes. Analyses of these mutations reveal that they increase the PI3K signal, stimulate downstream Akt signaling, promote growth factor-independent growth and increase cell invasion and metastasis. In this review, we summarize the PI3K/PTEN/AKT pathway genetic alterations in cancer and their potential clinical applications.     

Oncogenic PI3K and its role in cancer

PURPOSE OF REVIEW: The purpose of this review is to examine the contribution of the PI3K signaling pathway to the development of human tumors and to propose further studies to elucidate how to develop therapeutics for patients with mutations in this pathway. RECENT FINDINGS: More than 30% of various solid tumor types were recently found to contain mutations in PIK3CA, the catalytic subunit of PI3K. Further analysis of key genes in this pathway identified an additional eight genes altered in tumors. These were generally found to be mutated in a mutually exclusive manner, thus increasing the mutation frequency of the pathway to 40% in colorectal cancers and emphasizing the importance of the PI3K pathway in tumorigenesis. Functional analyses of PIK3CA mutations revealed that they increase its enzymatic activity, stimulate AKT signaling, allow growth factor-independent growth as well as increasing cell invasion and metastasis. SUMMARY: The PI3K signaling pathway is dysregulated by a variety of mechanisms in a large fraction of human tumors. Both mutational and functional analyses have shown that PIK3CA is an oncogene that plays an important role in tumor progression. Mutant members of the PI3K pathway, including PIK3CA, are good targets for therapeutic intervention because most of them are kinases, making them attractive for drug development. Gaining further insights into PIK3CA oncogenic mechanisms may produce new biomarkers and help the development of targeted therapeutics.     

HS-116, a novel phosphatidylinositol 3-kinase inhibitor induces apoptosis and suppresses angiogenesis of hepatocellular carcinoma through inhibition of the PI3K/AKT/mTOR pathway

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival of human cancers. As PI3K is active in many cancer patients, resulting in cancer development and progression, we developed an azaindole derivative, HS-116 as a novel PI3K inhibitor. This study aimed to clarify the anticancer effect of HS-116 in human hepatocellular carcinoma (HCC). To identify the effect of HS-116 on HCC cells, a PI3K assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and Western blotting were conducted. IC₅₀ of HS-116 for PI3Kα was 31 nM, and it effectively suppressed the phosphorylation of PI3K downstream factors such as AKT, mTOR, p70S6K, and 4EBP1. Also, HS-116 induced apoptosis by increasing the proportion of sub-G1 apoptotic cells from 1.8% to 35% and increasing the expressions of Bax, cleaved-caspase-3, and cleaved-PARP as well as decreasing the expression of Bcl-2. In addition, chromatin condensation and apoptotic bodies were detected in HS-116-treated HCC cells. Furthermore, HS-116 decreased protein expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), and inhibited the tube formation and migration of human umbilical vein endothelial cells (HUVECs). In vivo, the ability of mice to vascularize subcutaneously implanted Matrigel plugs was diminished when the mice were treated with HS-116. These results show that HS-116 inhibits the PI3K/AKT/mTOR pathway via apoptosis and anti-angiogenesis in HCC cells. We suggest that HS-116 may be an effective novel therapeutic candidate against HCC.     

PRMT9 promotes hepatocellular carcinoma invasion and metastasis via activating PI3K/Akt/GSK‐3β/Snail signaling

Protein arginine methyltransferases (PRMT) catalyze protein arginine methylation and play an important role in many biological processes. Aberrant PRMT expression in tumor cells has been documented in several common cancer types; however, its precise contribution to hepatocellular carcinoma (HCC) cell invasion and metastasis is not fully understood. In this study, we identified a new oncogene, PRMT9, whose overexpression strongly promotes HCC invasion and metastasis. PRMT9 expression was detected more frequently in HCC tissues than in adjacent noncancerous tissues. PRMT9 overexpression was significantly correlated with hepatitis B virus antigen (HBsAg) status, vascular invasion, poor tumor differentiation and advanced TNM stage. Patients with higher PRMT9 expression had a shorter survival time and higher recurrence rate. PRMT9 expression was an independent and significant risk factor for survival after curative resection. Functional studies demonstrated that PRMT9 increased HCC cell invasion and lung metastasis. Knocking down PRMT9 with short hairpin RNA (shRNA) inhibited HCC cell invasion. Further investigations found that PRMT9 increased cell migration and invasion through epithelial‐mesenchymal transition (EMT) by regulating Snail expression via activation of the PI3K/Akt/GSK‐3β/Snail signaling pathway. In clinical HCC samples, PRMT9 expression was positively associated with Snail expression and was negatively associated with E‐cadherin expression. In conclusion, our study demonstrated that PRMT9 is an oncogene that plays an important role in HCC invasion and metastasis through EMT by regulating Snail expression via activation of the PI3K/Akt/GSK‐3β/Snail signaling pathway. Thus, PRMT9 may serve as a candidate prognostic biomarker and a potential therapeutic target.     

Suppression of tumor proliferation and angiogenesis of hepatocellular carcinoma by HS-104, a novel phosphoinositide 3-kinase inhibitor

Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway frequently instigates tumorigenesis leading to hepatocellular carcinoma (HCC). We synthesized N-(5-(3-(3-methyl-1,2,4-oxadiazol-3-yl)imidazo[1,2-a]pyridin-6-yl)pyridin-3-yl)benzenesulfonamide (HS-104), a novel PI3K inhibitor, and investigated its in vitro anticancer effect and in vivo capacity in an animal xenograft model. The inhibition of cell growth by HS-104 revealed that it was effective against HCC cell lines. Also, the activation of the AKT/mTOR signal cascade was inhibited by HS-104 treatment in a dose dependent manner. Flow cytometry analysis showed an accumulation of HCC cells in the G2/M phase with concomitant loss of cells in the S phase. The apoptotic effect of HS-104 was accompanied by increased evidence of cleaved caspase-3 and PARP, as well as DNA fragmentation. In angiogenesis studies, HS-104 inhibited the tube formation of vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs), and suppressed microvessel sprouting from a rat aortic ring, ex vivo, and blood vessel formation in the Matrigel plug assay in mice. HS-104 inhibited the expression of the downstream proteins of PI3K including p-AKT, p-mTOR and p-p70S6K in VEGF-induced HUVECs. In the xenograft animal model, HS-104 significantly delayed tumor growth in a dose dependent manner and suppressed the expression of PCNA, CD34 and cleaved caspase-3 in tumor tissue. These studies show that HS-104 inhibited the PI3K/AKT/mTOR signaling pathway resulting in cell growth/angiogenesis inhibition and apoptosis induction. Therefore, HS-104 is considered as a novel drug candidate for the treatment of HCC.     

A virtual screen identified C96 as a novel inhibitor of phosphatidylinositol 3-kinase that displays potent preclinical activity against multiple myeloma in vitro and in vivo

The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is emerging as a promising therapeutic target for multiple myeloma (MM). In the present study, we performed a virtual screen against 800,000 of small molecule compounds by targeting PI3Kγ. C96, one of such compounds, inhibited PI3K activated by insulin-like growth factor-1 (IGF-1), but did not suppress IGF-1R activation. The cell-free assay revealed that C96 preferred to inhibit PI3Kα and δ, but was not active against AKT1, 2, 3 or mTOR. C96 inhibited PI3K activation in a time- and concentration-dependent manner. Consistent with its inhibition on PI3K/AKT, C96 downregulated the activation of mTOR, p70S6K, 4E-BP1, but did not suppress other kinases such as ERK and c-Src. Inhibition of the PI3K/AKT signaling pathway by C96 led to MM cell apoptosis which was demonstrated by Annexin V staining and activation of the pro-apoptotic signals. Furthermore, C96 displayed potent anti-myeloma activity in a MM xenograft model in nude mice. Oral administration of 100 mg/kg bodyweight almost fully suppressed tumor growth within 16 days, but without gross toxicity. Importantly, AKT activation was suppressed in tumor tissues from C96-treated mice, which was consistent with delayed tumor growth. Thus, we identified a novel PI3K inhibitor with a great potential for MM therapy.     

Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway.     

SIRT1 promotes tumorigenesis of hepatocellular carcinoma through PI3K/PTEN/AKT signaling

SIRT1 is the human orthologue of SIR2, a conserved NAD-dependent protein deacetylase that regulates longevity in yeast and in Caenorhabditis elegans. Overexpression of SIRT1 in cancer tissue, compared with normal tissue, has been demonstrated, suggesting that SIRT1 may act as a tumor promoter. The function of SIRT1 in liver cancer has not been elucidated. In the present study, SIRT1 re-expression or knockdown was induced in hepatoma cell lines and liver normal cell lines. Our study demonstrated that overexpression of SIRT1 promoted mitotic entry of liver cells, cell growth and proliferation and inhibited apoptosis. The apoptosis involved caspase-3 and caspase-7, and was related to the PTEN/PI3K/AKT signaling pathway. The results demonstrate that SIRT1 promotes tumorigenesis of hepatocellular carcinoma (HCC) through the PTEN/PI3K/AKT signaling pathway. SIRT1 may serve as a novel target for selective killing of cancer versus normal liver cells.     

Targeting the PI3K/AKT/mTOR pathway in biliary tract cancers: A review of current evidences and future perspectives

Biliary tract cancers (BTCs) are a group of invasive neoplasms, with increasing incidence and dismal prognosis. In advanced disease, the standard of care is represented by first-line chemotherapy with cisplatin and gemcitabine. In subsequent lines, no clear recommendations are currently available, highlighting the need for novel therapeutic approaches.The PI3K/AKT/mTOR pathway is a core regulator of cell metabolism, growth and survival, and is involved in BTCs carcinogenesis and progression. Mutations, gene copy number alterations and aberrant protein phosphorylation of PI3K, AKT, mTOR and PTEN have been thoroughly described in BTCs and correlate with poor survival outcomes.Several pre-clinical evidences state the efficacy of PI3K/AKT/mTOR pathway inhibitors in BTCs, both in vitro and in vivo. In the clinical setting, initial studies with rapamycin analogs have shown interesting activity with an acceptable toxicity profile. Novel strategies evaluating AKT and PI3K inhibitors have risen serious safety concerns, pointing out the need for improved patient selection and increased target specificity for the clinical development of these agents, both alone and in combination with chemotherapy.This review extensively describes the role of the PI3K/AKT/mTOR pathway in BTCs and examines the rationale of its targeting in these tumors, with particular focus on clinical activity, toxicities and perspectives on further development of PI3K/AKT/mTOR pathway inhibitors.     

A specific role for AKT3 in the genesis of ovarian cancer through modulation of G(2)-M phase transition

Ovarian cancer is the major cause of death from gynecological malignancy, and there is an urgent need for new therapeutic targets. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been strongly implicated in the genesis of ovarian cancer. However, to identify and evaluate potential targets for therapeutic intervention, it is critical to understand the mechanism by which the PI3K/AKT pathway facilitates ovarian carcinogenesis. Here, we show that AKT3 is highly expressed in 19 of 92 primary ovarian tumors. Strikingly, purified AKT3 exhibited up to 10-fold higher specific activity than AKT1, potentially amplifying the effects of AKT3 overexpression. Consistent with this finding, AKT3 levels in a range of ovarian cancer cell lines correlated with total AKT activity and proliferation rates, implicating AKT3 as a key mediator of ovarian oncogenesis. Specific silencing of AKT3 using short hairpin RNA markedly inhibited proliferation of the two cell lines with highest AKT3 expression and total AKT activity, OVCA429 and DOV13, by slowing G(2)-M phase transition. These findings are consistent with AKT3 playing a key role in the genesis of at least one subset of ovarian cancers.     

A novel PI3K inhibitor PIK-C98 displays potent preclinical activity against multiple myeloma

Recent clinical trials have demonstrated targeting PI3K pathway is a promising strategy for the treatment of blood cancers. To identify novel PI3K inhibitors, we performed a high throughput virtual screen and identified several novel small molecule compounds, including PIK-C98 (C98). The cell-free enzymatic studies showed that C98 inhibited all class I PI3Ks at nano- or low micromolar concentrations but had no effects on AKT or mTOR activity. Molecular docking analysis revealed that C98 interfered with the ATP-binding pockets of PI3Ks by forming H-bonds and arene-H interactions with specific amino acid residues. The cellular assays demonstrated that C98 specifically inhibited PI3K/AKT/mTOR signaling pathway, but had no effects on other kinases and proteins including IGF-1R, ERK, p38, c-Src, PTEN, and STAT3. Inhibition of PI3K by C98 led to myeloma cell apoptosis. Furthermore, oral administration of C98 delayed tumor growth in two independent human myeloma xenograft models in nude mice but did not show overt toxicity. Pharmacokinetic analyses showed that C98 was well penetrated into myeloma tumors. Therefore, through a high throughput virtual screen we identified a novel PI3K inhibitor that is orally active against multiple myeloma with great potential for further development.     

Targeting PI3K and RAD51 in Barrett's adenocarcinoma: impact on DNA damage checkpoints, expression profile and tumor growth

Phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog 1 (AKT) signaling in cancer is implicated in various survival pathways including regulation of recombinase (RAD51). In this study, we evaluated PI3K and RAD51 as targets in Barrett's adenocarcinoma (BAC) cells both in vitro and in vivo. BAC cell lines (OE19, OE33, and FLO-1) were cultured in the presence of PI3K inhibitor (wortmannin) and the impact on growth and expression of AKT, phosphorylated-AKT (P-AKT), and RAD51 was determined. Wortmannin induced growth arrest and apoptosis in two BAC cell lines (OE33 and OE19), which had relatively higher expression of AKT. FLO-1 cells, with lower AKT expression, were less sensitive to treatment and investigated further. In FLO-1 cells, wortmannin suppressed ataxia telangiectasia and Rad3-related protein (ATR)-checkpoint kinase 1 (CHK1)-mediated checkpoint and multiple DNA repair genes, whereas RAD51 and CHK2 were not affected. Western blotting confirmed that RAD51 was suppressed by wortmannin in OE33 and OE19 cells, but not in FLO-1 cells. Suppression of RAD51 in FLO-1 cells down-regulated the expression of CHK2 and CHK1, and reduced the proliferative potential. Finally, the suppression of RAD51 in FLO-1 cells, significantly increased the anticancer activity of wortmannin in these cells, both in vitro and in vivo. We show that PI3K signaling and hsRAD51, through distinct roles in DNA damage response and repair pathways, provide survival advantage to BAC cells. In cells with inherent low expression of AKT, RAD51 is unaffected by PI3K suppression and provides an additional survival pathway. Simultaneous suppression of PI3K and RAD51, especially in cells with lower AKT expression, can significantly reduce their proliferative potential.     

Metformin and trametinib have synergistic effects on cell viability and tumor growth in NRAS mutant cancer

Attempts to directly block the mutant neuroblastoma rat sarcoma oncogene (NRAS) protein, a driving mutation in many cancer types, have been unsuccessful. Current treatments focus on inhibition of different components of NRAS'' two main downstream cascades: PI3K/AKT/mTOR and MAPK. Here we test a novel dual therapy combination of metformin and trametinib on a panel of 16 NRAS mutant cell lines, including melanoma cells, melanoma cells with acquired trametinib resistance, lung cancer and neuroblastoma cells. We show that both of the main downstream cascades of NRAS can be blocked by this combination: metformin indirectly inhibits the PI3K/AKT/mTOR pathway and trametinib directly impedes the MAPK pathway. This dual therapy synergistically reduced cell viability in vitro and xenograft tumor growth in vivo. We conclude that metformin and trametinib combinations are effective in preclinical models and may be a possible option for treatment of NRAS mutant cancers.