The transcription factor SOX2 is an essential regulator of pluripotent stem cells and promotes development and maintenance of squamous epithelia. We previously reported that SOX2 is an oncogene and subject to highly recurrent genomic amplification in squamous cell carcinomas (SCCs). Here, we have further characterized the function of SOX2 in SCC. Using ChIP-seq analysis, we compared SOX2-regulated gene profiles in multiple SCC cell lines to ES cell profiles and determined that SOX2 binds to distinct genomic loci in SCCs. In SCCs, SOX2 preferentially interacts with the transcription factor p63, as opposed to the transcription factor OCT4, which is the preferred SOX2 binding partner in ES cells. SOX2 and p63 exhibited overlapping genomic occupancy at a large number of loci in SCCs; however, coordinate binding of SOX2 and p63 was absent in ES cells. We further demonstrated that SOX2 and p63 jointly regulate gene expression, including the oncogene ETV4, which was essential for SOX2-amplified SCC cell survival. Together, these findings demonstrate that the action of SOX2 in SCC differs substantially from its role in pluripotency. The identification of the SCC-associated interaction between SOX2 and p63 will enable deeper characterization the downstream targets of this interaction in SCC and normal squamous epithelial physiology.

Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasomemediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.FOXO (forkhead O) proteins belong to the family of forkhead transcriptional factors, which are characterized by a conserved DNA binding domain termed the "Forkhead box" (1). Mammalian FOXO factors include FOXO1 (previously known as FKHR), FOXO3A (previously known as FKHRL1), FOXO4 (previously known as AFX), and FOXO6. These factors control the expression of a variety of genes that regulate essential cellular processes, such as cell cycle (2-4), apoptosis (5), oxidative stress (6, 7), atrophy (8), energy homeostasis, and glucose metabolism (9, 10). Whole organism studies in worms and flies show that FOXO factors have conserved ability to increase the organismal longevity (11). Genetic and functional analysis identifies FOXO1 as a tumor suppressor in the prostate (12). Knock-out studies show that mammalian FOXO factors act redundantly to suppress tumorigenesis in a lineage-specific fashion (13) and to maintain the long term regenerative potential of hematopoietic stem cells by regulating genes involved in the cellular response to physiological oxidative stresses (14).The transcription of FOXO factors is regulated by posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. Multiple kinases, including AKT (15, 16), serum-and glucocorticoid-induced kinase (17), casein kinase 1 (18), mammalian Ste20-like kinase 1 (19), I B kinase (20), and cyclin-dependent kinase 2 (21), catalyze FOXO phosphorylation and often promote FOXO nuclear exportation. In response to insulin and growth factors, FOXO1 and FOXO3A are ubiquitinated and degraded by the proteasome pathway after phosphorylation at known AKT sites (15,22,23). Acetyltransferases, p300 (24) and CBP (25), and SIRT1 deacetylase (26, 27) regulate the activity of FOXO through acetylation/deacetylation. The role of FOXO acetylation is controversial, but it could affect their nuclear retention (28), phosphorylation (25), and ubiquitination-medi...

FoxO (mammalian forkhead subclass O) proteins are transcription factors acting downstream of the PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor. Their activity is negatively regulated by AKT-mediated phosphorylation. Our previous studies showed that the transcriptional activity of the androgen receptor (AR) was inhibited by PTEN in an AKT-sensitive manner. Here, we report the repression of the activity of the full-length AR and its N-terminal domain by FoxO1 and the participation of FoxO1 in AR inhibition by PTEN. Ectopic expression of active FoxO1 decreased the transcriptional activity of AR as well as androgen-induced cell proliferation and production of prostate-specific antigen. FoxO1 knock down by RNA interference increased the transcriptional activity of the AR in PTEN-intact cells and relieved its inhibition by ectopic PTEN in PTEN-null cells. Mutational analysis revealed that FoxO1 fragment 150-655, which contains the forkhead box and C-terminal activation domain, was required for AR inhibition. Mammalian two-hybrid and glutathione-S-transferase pull-down assays demonstrated that the inhibition of AR activity by PTEN through FoxO1 involved the interference of androgen-induced interaction of the N- and C-termini of the AR and the recruitment of the p160 coactivators to its N terminus and to the androgen response elements of natural AR target genes. These studies reveal new mechanisms for the inhibition of AR activity by PTEN-FoxO axis and establish FoxO proteins as important nuclear factors that mediate the mutual antagonism between AR and PTEN tumor suppressor in prostate cancer cells.

Ultrasmall gold nanoclusters (AuNCs) are emerging as promising luminescent nanoprobes for bioimaging due to their fantastic photoluminescence (PL) and renal-clearable ability. However, it remains a great challenge to design them for in vivo sensitive molecular imaging in desired tissues. Herein, we have developed a strategy to tailor the PL and biofate of near-infrared II (NIR-II)-emitting AuNCs via ligand anchoring for improved bioimaging. By optimizing the ligand types in AuNCs and using Er3+-doped lanthanide (Ln) nanoparticles as models, core–satellite Ln@AuNCs assemblies were rationally constructed, which enabled 2.5-fold PL enhancement of AuNCs at 1100 nm and prolonged blood circulation compared to AuNCs. Significantly, Ln@AuNCs with dual intense NIR-II PL (from AuNCs and Er3+) can effectively accumulate in the liver for ratiometric NIR-II imaging of H2S, facilitated by H2S-mediated selective PL quenching of AuNCs. We have then demonstrated the real-time imaging evaluation of liver delivery efficacy and dynamics of two H2S prodrugs. This shows a paradigm to visualize liver H2S delivery and its prodrug screening in vivo. Note that Ln@AuNCs are body-clearable via the hepatobiliary excretion pathway, thus reducing potential long-term toxicity. Such findings may propel the engineering of AuNC nanoprobes for advancing in vivo bioimaging analysis.