Identification of VPS34-PI(3)P-FEN1-mediated DNA repair pathway as a potential drug target to overcome chemoresistance.

Intrinsic or acquired chemoresistance represents a major obstacle in cancer treatment. Multiple mechanisms can contribute to cancer cells' resistance to chemotherapy. Among them, an aberrantly strengthened DNA repair mechanism is responsible for a large proportion of drug resistance to alkylating agents and radiation therapy. In cancer cells, damping overactivated DNA repair system can overcome survival advantages conferred by chromosomal translocations or mutations and lead to cytostatic effects or cytotoxic. Therefore, selectively targeting DNA repair system in cancer cells holds promise for overcoming chemoresistance. In this study, we revealed that the endonuclease Flap Endonuclease 1 (FEN1), essential for DNA replication and repair, directly interacts with phosphatidylinositol 3-phosphate [PI(3)P], and FEN1-R378 is the primary PI(3)P-binding site. PI(3)P-binding deficient FEN1 mutant (FEN1-R378A) cells exhibited abnormal chromosomal structures and were hypersensitized to DNA damage. The PI(3)P-mediated FEN1 functionality was essential for repairing DNA damages caused by multiple mechanisms. Furthermore, VPS34, the major PI(3)P synthesizing enzyme, was negatively associated with patients' survival in various cancer types, and VPS34 inhibitors significantly sensitized chemoresistant cancer cells to genotoxic agents. These findings open up an avenue for counteracting chemoresistance by targeting VPS34-PI(3)P-mediated DNA repair pathway, and call for assessing the efficacy of this strategy in patients suffering from chemoresistance-mediated cancer recurrence in clinical trials.

Translational and post-translational control of human naïve versus primed pluripotency.

Deciphering the regulatory network for human naive and primed pluripotency is of fundamental theoretical and applicable significance. Here, by combining quantitative proteomics, phosphoproteomics, and acetylproteomics analyses, we revealed RNA processing and translation as the most differentially regulated processes between naive and primed human embryonic stem cells (hESCs). Although glycolytic primed hESCs rely predominantly on the eukaryotic initiation factor 4E (eIF4E)-mediated cap-dependent pathway for protein translation, naive hESCs with reduced mammalian target of rapamycin complex (mTORC1) activity are more tolerant to eIF4E inhibition, and their bivalent metabolism allows for translating selective mRNAs via both eIF4E-dependent and eIF4E-independent/eIF4A2-dependent pathways to form a more compact naive proteome. Globally up-regulated proteostasis and down-regulated post-translational modifications help to further refine the naive proteome that is compatible with the more rapid cycling of naive hESCs, where CDK1 plays an indispensable coordinative role. These findings may assist in better understanding the unrestricted lineage potential of naive hESCs and in further optimizing conditions for future clinical applications.

DNA repair proteins cooperate with SOX2 in regulating the transition of human embryonic stem cells to neural progenitor cells.

SOX2, a well-established pluripotency factor supporting the self-renewal of pluripotent stem cells (PSCs), is also a crucial factor for maintaining the properties and functionalities of neural progenitor cells (NPCs). It regulates the transcription of target genes by forming complexes with its partner factors, but systematic comparison of SOX2 binding partners in human PSCs versus NPCs is lacking. Here, by deciphering and comparing the SOX2-protein interactomes in human embryonic stem cells (hESCs) versus the NPCs derived from them, we identified 23 proteins with high reproducibility that are most differentially associated with SOX2, of which 9 are DNA repair proteins (PARP1, PARP2, PRKDC, XRCC1, XRCC5, XRCC6, RPA1, LIG3, DDB1). Genetic knocking-down or pharmacological inhibiting two of the DNA repair proteins (PARP1 and PRKDC) significantly up-regulated certain NPC or ectodermal biomarkers that are transcriptionally-suppressed by the SOX2/DNA repair protein complexes. These findings point to a crucial role of DNA repair proteins in pluripotent state transition and neural induction.

LSD1-mediated demethylation of OCT4 safeguards pluripotent stem cells by maintaining the transcription of PORE-motif-containing genes.

Reversible lysine methylation is essential for regulating histones and emerges to critically regulate non-histone proteins as well. Here we show that the master transcription factor OCT4 in pluripotent stem cells (PSCs) was methylated at multiple lysine residues. LSD1 that is highly expressed in PSCs can directly interact with and demethylate OCT4 at lysine 222 (K222) in the flexible linker region. Reduced LSD1 activity led to the methylation of OCT4-K222 that diminished the differentiation potential of PSCs while facilitating proteasome-independent degradation of OCT4 proteins. Furthermore, site-specifically replacing K222 with phenylalanine to mimic the constitutively methylated lysine promoted the 'locked-in' mode engagement of the OCT4 PORE-homodimers that tightly bind to and block the transcription of multiple PORE-motif-containing target genes regulating cell fate determination and cell junction organization, and thereby reducing the pluripotency of PSCs. Thus, LSD1-mediated demethylation of OCT4 plays a crucial role in restricting the 'locked-in' mode binding of OCT4 PORE-homodimers to the PORE-motif-containing genes and thereby maintaining their transcription to safeguard the pluripotency of PSCs.

Combined inhibition of JAK1/2 and DNMT1 by newly identified small-molecule compounds synergistically suppresses the survival and proliferation of cervical cancer cells.

Despite substantial advances in treating cervical cancer (CC) with surgery, radiation and chemotherapy, patients with advanced CC still have poor prognosis and significantly variable clinical outcomes due to tumor recurrence and metastasis. Therefore, to develop more efficacious and specific treatments for CC remains an unmet clinical need. In this study, by virtual screening the SPECS database, we identified multiple novel JAK inhibitor candidates and validated their antitumor drug efficacies that were particularly high against CC cell lines. AH057, the best JAK inhibitor identified, effectively blocked the JAK/STAT pathways by directly inhibiting JAK1/2 kinase activities, and led to compromised cell proliferation and invasion, increased apoptosis, arrested cell cycles, and impaired tumor progression in vitro and in vivo. Next, by screening the Selleck chemical library, we identified SGI-1027, a DNMT1 inhibitor, as the compound that displayed the highest synergy with AH057. By acting on a same set of downstream effector molecules that are dually controlled by JAK1/2 and DNMT1, the combination of AH057 with SGI-1027 potently and synergistically impaired CC cell propagation via dramatically increasing apoptotic cell death and cell-cycle arrest. These findings establish a preclinical proof of concept for combating CC by dual targeting of JAK1/2 and DNMT1, and provide support for launching a clinical trial to evaluate the efficacy and safety of this drug combination in patients with CC and other malignant tumors.

Endogenous authentic OCT4A proteins directly regulate FOS/AP-1 transcription in somatic cancer cells.

OCT4A is well established as a master transcription factor for pluripotent stem cell (PSC) self-renewal and a pioneer factor for initiating somatic cell reprogramming, yet its presence and functionality in somatic cancer cells remain controversial and obscure. By combining the CRISPR-Cas9-based gene editing with highly specific PCR assays, highly sensitive immunoassays, and mass spectrometry, we provide unequivocal evidence here that full-length authentic OCT4A transcripts and proteins were both present in somatic cancer cells, and OCT4A proteins were heterogeneously expressed in the whole cell population and when expressed, they are predominantly localized in cell nucleus. Despite their extremely low abundance (approximately three orders of magnitude lower than in PSCs), OCT4A proteins bound to the promoter/enhancer regions of the AP-1 transcription factor subunit c-FOS gene and critically regulated its transcription. Knocking out OCT4A in somatic cancer cells led to dramatic reduction of the c-FOS protein level, aberrant AP-1 signaling, dampened self-renewal capacity, deficient cell migration that were associated with cell growth retardation in vitro and in vivo, and their enhanced sensitivity to anticancer drugs. Taken together, we resolve the long-standing controversy and uncertainty in the field, and reveal a fundamental role of OCT4A protein in regulating FOS/AP-1 signaling-centered genes that mediate the adhesion, migration, and propagation of somatic cancer cells.

Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells.

AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin + Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells.

Site-specific Disruption of the Oct4/Sox2 Protein Interaction Reveals Coordinated Mesendodermal Differentiation and the Epithelial-Mesenchymal Transition.

Although the Oct4/Sox2 complex is crucial for maintaining the pluripotency of stem cells, the molecular basis underlying its regulation during lineage-specific differentiation remains unknown. Here, we revealed that the highly conserved Oct4/Lys-156 is important for maintaining the stability of the Oct4 protein and the intermolecular salt bridge between Oct4/Lys-151 and Sox2/Asp-107 that contributes to the Oct4/Sox2 interaction. Post-translational modifications at Lys-156 and K156N, a somatic mutation detected in bladder cancer patients, both impaired the Lys-151-Asp-107 salt bridge and the Oct4/Sox2 interaction. When produced as a recombinant protein or overexpressed in pluripotent stem cells, Oct4/K156N, with reduced binding to Sox2, significantly down-regulated the stemness genes that are cooperatively controlled by the Oct4/Sox2 complex and specifically up-regulated the mesendodermal genes and the SNAIL family genes that promote the epithelial-mesenchymal transition. Thus, we conclude that Oct4/Lys-156-modulated Oct4/Sox2 interaction coordinately controls the epithelial-mesenchymal transition and mesendoderm specification induced by specific differentiation signals.

Tryptophan derivatives regulate the transcription of Oct4 in stem-like cancer cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to environmental toxicants, is increasingly recognized as a key player in embryogenesis and tumorigenesis. Here we show that a variety of tryptophan derivatives that act as endogenous AhR ligands can affect the transcription level of the master pluripotency factor Oct4. Among them, ITE enhances the binding of the AhR to the promoter of Oct4 and suppresses its transcription. Reduction of endogenous ITE levels in cancer cells by tryptophan deprivation or hypoxia leads to Oct4 elevation, which can be reverted by administration with synthetic ITE. Consequently, synthetic ITE induces the differentiation of stem-like cancer cells and reduces their tumorigenic potential in both subcutaneous and orthotopic xenograft tumour models. Thus, our results reveal a role of tryptophan derivatives and the AhR signalling pathway in regulating cancer cell stemness and open a new therapeutic avenue to target stem-like cancer cells.

14-3-3ζ reduces DNA damage by interacting with and stabilizing proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a processivity factor of DNA replication which plays critical roles in the regulation of DNA replication and repair. In this study, we show that PCNA interacts directly in vitro and in cells with 14-3-3ζ, an adaptor protein that regulates cell growth and response to DNA damage in eukaryotes. The interaction is mediated by at least two PCNA-binding sites on 14-3-3ζ, one of which is a novel non-canonical PIP (PCNA interacting protein) box. We find that DNA damages induced by UVC irradiation and MMS (methyl methanesulfonate) can enhance both the interaction of these two proteins and their co-localization with chromatin. Functional analyses suggest that 14-3-3ζ stabilizes PCNA possibly by regulating its ubiquitination, which impacts on DNA damage repair and cell viability.

A cell-free system toward deciphering the post-translational modification barcodes of Oct4 in different cellular contexts.

The octamer-binding transcription factor 4 (Oct4) is essential for maintaining the self-renewal and pluripotency of embryonic stem cells (ESCs). Post-translational modifications (PTMs) of Oct4 critically control its structure, function and intracellular localization. However, determination of Oct4 PTM profiles has largely been restricted by the quantity and purity of the Oct4 protein samples required for mass spectrometric analyses. In this study, by incubating the Escherichia coli-derived His-tagged Oct4 proteins with the whole cell lysates of a variety of human cells followed by retrieving the reacted Oct4 proteins with the Ni-NTA beads, we developed a labor- and cost-effective in vitro PTM method that allowed for mass spectrometric determination of the phosphorylation profiles of Oct4 proteins exposed to various cell-free systems. A number of Oct4 phosphorylation sites that were commonly present in all the cell-free systems or specifically present in a particular cellular context were identified, indicating that Oct4 is controlled by both common and distinct PTM regulatory pathways. Our work provided a proof-of-concept that such a cell-free system-based in vitro PTM approach can be applied to systematically map out the physiologically-relevant PTM sites in Oct4 proteins, which opened up an avenue to fully decipher the Oct4 PTM barcodes in various cellular contexts.