TGFβ induces stemness through non-canonical AKT-FOXO3a axis in oral squamous cell carcinoma.

FOXO3a has been widely regarded as a tumor suppressor. It also plays a paradoxical role in regulating the cancer stem cells (CSCs), responsible for tumor-initiation, chemo-resistance, and recurrence in various solid tumors, including oral squamous cell carcinoma (OSCC). This study aims to uncover the role of FOXO3a and its importance for a non-canonical pathway of TGFβ in regulating the OSCC stemness. We identified FOXO3a expression in OSCC tissues and cell lines using immunohistochemistry and western blot. The correlation between FOXO3a and stemness was evaluated. Stable cell lines with differential expression of FOXO3a were constructed using lentiviruses. The effects of FOXO3a on stem-cell like properties in OSCC was further evaluated in vitro and in vivo. We also explored the effect of TGFβ on FOXO3a with respect to its expression and function. Our findings suggest that FOXO3a was widely expressed and negatively correlated with the stemness in OSCC. This regulation can be abolished by TGFβ through phosphorylation, nuclear exclusion, and degradation in the non-Smad pathway. We also observed that non-Smad AKT-FOXO3a axis is essential to regulate stemness of CSCs by TGFβ. TGFβ induces stemness through non-canonical AKT-FOXO3a axis in OSCC. Our study provides a foundation to understand the mechanism of CSCs and a possible therapeutic target to eliminate CSCs.

Li K ，Yang L ，Li J ，Guan C ，Zhang S ，Lao X ，Ouyang D ，Zheng G ，Gao S ，Wang D ，Liang Y ，Liao G ... -  《EBioMedicine》

Akt-FOXO3a signaling axis dysregulation in human oral squamous cell carcinoma and potent efficacy of FOXO3a-targeted gene therapy.

Phosphoinositide-3-kinase (PI3K)/Akt pathway has been shown to be activated in oral squamous cell carcinoma (OSCC). Activation of Akt suppresses FOXO transcription factor-mediated growth arrest and apoptosis in various cancers. We investigated FOXO3a and phosphor(p)-Akt expression and potential efficacy of FOXO3a-targeted gene therapy in OSCC. Low expression of FOXO3a was negatively associated with overexpression of p-Akt and histological grade using immunohistochemistry. Akt-FOXO3a axis was also examined by detection of FOXO3a expression after induction or inhibition of Akt activity in Tca8113 OSCC cells. Transfection of a constitutively active form of FOXO3a (FOXO3a(3A)) in OSCC cells induced significant G₁-phase arrest and apoptosis as compared with control and transfection of a wild-type FOXO3a (FOXO3a(WT)). Stable FOXO3a(3A) transfectant OSCC cells also revealed the most potent growth inhibition effect in vivo. Furthermore, the downstream effects of FOXO3a activation were found to be inhibition of CDK4/6 and cyclin D1, and accumulation of p27 and Bim. We also found that transcription of FOXO1 and FOXO4 were stimulated by FOXO3a. Our results suggest that FOXO3a activity may be important in tumorigenesis and development of OSCC. Akt-FOXO3a axis inhibition-mediated constitutively active FOXO3a induces significant growth inhibition and FOXO3a activation may present a potent target-based therapeutic strategy for OSCC therapy.

Fang L ，Wang H ，Zhou L ，Yu D ... -  《-》

ZNF677 inhibits oral squamous cell carcinoma growth and tumor stemness by regulating FOXO3a.

Oral squamous cell carcinoma (OSCC) is a common cancer with an increasing incidence worldwide. Zinc-finger proteins 677 (ZNF677) is involved in the progression and methylation of various cancers, but its role and mechanism in OSCC remain indeterminate. The expression of ZNF677 was analyzed by online database and immunohistochemistry, while the methylation level of ZNF677 was determined by the methylation-specific PCR. The role and mechanism of ZNF677 in the tumor cell growth, migration, invasion and stemness were addressed by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation, Transwell, wound-healing, sphere‑formation, and western blot assays. In addition, its function was also investigated in a xenografted mice model. The results showed that ZNF677 was lowly expressed in OSCC with a hypermethylation level, which predicted poor overall survival in patients with HNSC. Upregulation of ZNF677 reduced the cell viability, Edu positive cells, numbers of invasion cells, the migration ability, numbers of spheres formation and the expression of proliferation, migration and stemness related proteins in CAL-27 and SCC25 cells. Mechanically, the relative levels of p-AKT/AKT were decreased and the levels of p-FOXO3a/FOXO3a were increased in both cells overexpressed with ZNF677, which were reversed by the SC79 treatment. Moreover, interference of FOXO3a recovered the suppressive effects of ZNF677 overexpression on cell proliferation, migration, invasion and stemness of OSCC cells. Furthermore, overexpression of ZNF677 reduced the tumor volume and weight, and the relative protein level of p-AKT/AKT with an increased level of p-FOXO3a/FOXO3a, and improved pathological symptoms in vivo. Collectively, ZNF677 suppressed OSCC cells growth, migration, invasion and stemness through inhibiting AKT/FOXO3a pathway.

Zhang Z ，Zhang Y ，Hu X ，Chen Y ，Zhuang L ，Zhang S ... -  《Human Cell》

Pitavastatin induces apoptosis in oral squamous cell carcinoma through activation of FOXO3a.

Statins are a class of lipid-lowering drugs that have recently been used in drug repositioning in the treatment of human cancer. However, the underlying mechanism of statin-induced cancer cell death has not been clearly defined. In the present study, we evaluated the anticancer effect of pitavastatin on oral squamous cell carcinoma (OSCC), SCC15 and SCC4 cells and found that FOXO3a might be a direct target in pitavastatin-induced cancer cell death. Our data revealed that pitavastatin selectively suppressed cell viability and induced intrinsic apoptosis in a FOXO3a-dependent manner in SCC15 cells while no effect was observed in SCC4 cells. Notably, treatment with pitavastatin in SCC15 cells induced the nuclear translocation of FOXO3a via dual regulation of two upstream kinases, AMPK and Akt, resulting in the up-regulation of PUMA, a transcriptional target gene of FOXO3a. Furthermore, our data revealed that FOXO3a-mediated PUMA induction plays a role in pitavastatin-induced intrinsic apoptosis in SCC15 cells. Taken together, our findings suggest that pitavastatin activates the FOXO3a/PUMA apoptotic axis by regulation of nuclear translocation of FOXO3a via Akt/FOXO3a or AMPK/FOXO3a signalling. Therefore, these findings might help to elucidate the underlying mechanism of the anticancer effects of pitavastatin on OSCC.

Lee N ，Tilija Pun N ，Jang WJ ，Bae JW ，Jeong CH ... -  《-》

FOXO3a reactivation mediates the synergistic cytotoxic effects of rapamycin and cisplatin in oral squamous cell carcinoma cells.

FOXO3a, a well-known transcriptional regulator, controls a wide spectrum of biological processes. The phosphoinositide-3-kinase (PI3K)/Akt signaling pathway inactivates FOXO3a via phosphorylation-induced nuclear exclusion and degradation. A loss or gain of FOXO3a activity has been correlated with efficiency of chemotherapies in various cancers including oral squamous cell carcinoma (OSCC). Therefore, in the current study, we have investigated the FOXO3a activity modulating and antitumor effects of rapamycin and cisplatin in OSCC cells. Cisplatin inhibited proliferation and induced apoptosis in a dose-dependent way in OSCC Tca8113 cells. Rapamycin alone had no effect on cell proliferation and apoptosis. Rapamycin downregulated the expression of S-phase kinase associated protein-2 (Skp2) and increased the FOXO3a protein stability but induced the upregulation of feedback Akt activation-mediated FOXO3a phosphorylation. Cisplatin decreased the phosphorylation of FOXO3a via Akt inhibition. Rapamycin combined with cisplatin as its feedback Akt activation inhibitor revealed the most dramatic FOXO3a nuclear localization and reactivation with the prevention of its feedback loop and exposed significant synergistic effects of decreased cell proliferation and increased apoptosis in vitro and decreased tumor size in vivo. Furthermore, the downstream effects of FOXO3a reactivation were found to be accumulation of p27 and Bim. In conclusion, rapamycin/cisplatin combination therapy boosts synergistic antitumor effects through the significant FOXO3a reactivation in OSCC cells. These results may represent a novel mechanism by which rapamycin/cisplatin combination therapy proves to be a potent molecular-targeted strategy for OSCC.

Fang L ，Wang H ，Zhou L ，Yu D ... -  《-》