Feng Yin

Pluripotent Stem Cell Protein Sox2 Confers Sensitivity to LSD1 Inhibition in Cancer Cells

Gene amplification of Sox2 at 3q26.33 is a common event in squamous cell carcinomas (SCCs) of the lung and esophagus, as well as several other cancers. Here, we show that the expression of LSD1/KDM1 histone demethylase is significantly elevated in Sox2-expressing lung SCCs. LSD1-specific inhibitors selectively impair the growth of Sox2-expressing lung SCCs, but not that of Sox2-negative cells. Sox2 expression is associated with sensitivity to LSD1 inhibition in lung, breast, ovarian, and other carcinoma cells. Inactivation of LSD1 reduces Sox2 expression, promotes G1 cell-cycle arrest, and induces genes for differentiation by selectively modulating the methylation states of histone H3 at lysines 4 (H3K4) and 9 (H3K9). Reduction of Sox2 further suppresses Sox2-dependent lineage-survival oncogenic potential, elevates trimethylation of histone H3 at lysine 27 (H3K27) and enhances growth arrest and cellular differentiation. Our studies suggest that LSD1 serves as a selective epigenetic target for therapy in Sox2-expressing cancers.

A Light-Driven Therapy of Pancreatic Adenocarcinoma Using Gold Nanorods-Based Nanocarriers for Co-Delivery of Doxorubicin and siRNA.

In this work, we report the engineering of polyelectrolyte polymers coated Gold nanorods (AuNRs)-based nanocarriers that are capable of co-delivering small interfering RNA (siRNA) and an anticancer drug doxorubicin (DOX) to Panc-1 cancer cells for combination of both chemo- and siRNA-mediated mutant K-Ras gene silencing therapy. Superior anticancer efficacy was observed through synergistic combination of promoted siRNA and DOX release upon irradiating the nanoplex formulation with 665 nm light. Our antitumor study shows that the synergistic effect of AuNRs nanoplex formulation with 665 nm light treatment is able to inhibit the in vivo tumor volume growth rate by 90%. The antitumor effect is contributed from the inactivation of K-Ras gene and thereby causing a profound synthesis (S) phase arrest in treated Panc-1 cells. Our study shows that the percentage of Panc-1 cells treated by nanoplex formulation with S phase is determined to be 35% and it is 17% much higher than that of Panc-1 cells without any treatments. The developed nanotherapy formulation here, that combines chemotherapy, RNA silencing and NIR window light-mediated therapy, will be seen to be the next natural step to be taken in the clinical research for improving the therapeutic outcomes of the pancreatic adenocarcinoma treatment.

LSD1 Regulates Pluripotency of Embryonic Stem/Carcinoma Cells through Histone Deacetylase 1-Mediated Deacetylation of Histone H4 at Lysine 16

ABSTRACT LSD1 is essential for the maintenance of pluripotency of embryonic stem (ES) or embryonic carcinoma/teratocarcinoma (EC) cells. We have previously developed novel LSD1 inhibitors that selectively inhibit ES/EC cells. However, the critical targets of LSD1 remain unclear. Here, we found that LSD1 interacts with histone deacetylase 1 (HDAC1) to regulate the proliferation of ES/EC cells through acetylation of histone H4 at lysine 16 (H4K16), which we show is a critical substrate of HDAC1. The LSD1 demethylase and HDAC1 deacetylase activities were both inactivated if one of them in the complex was chemically inhibited in ES/EC cells or in reconstituted protein complexes. Loss of HDAC1 phenocopied the selective growth-inhibitory effects and increased the levels of H3K4 methylation and H4K16 acetylation of LSD1 inactivation on ES/EC cells. Reduction of acetylated H4K16 by ablation of the acetyltransferase males absent on the first (MOF) is sufficient to rescue the growth inhibition induced by LSD1 inactivation. While LSD1 or HDAC1 inactivation caused the downregulation of Sox2 and Oct4 and induction of differentiation genes, such as FOXA2 or BMP2, depletion of MOF restored the levels of Sox2, Oct4, and FoxA2 in LSD1-deficient cells. Our studies reveal a novel mechanism by which LSD1 acts through the HDAC1- and MOF-mediated regulation of H4K16 acetylation to maintain the pluripotency of ES/EC cells.

SiRNA Delivery with PEGylated Graphene Oxide Nanosheets for Combined Photothermal and Genetherapy for Pancreatic Cancer.

Since the successful exfoliation of graphene from graphite in 2004, graphene and graphene oxide (GO) have been considered the most promising two-dimensional (2D) nanomaterials with distinguished physical and chemical characteristics and have attracted great attention in many different fields. Graphene oxide is well-known for its distinct physiochemical properties and shows only minimal cytotoxicity compared to carbon nanotubes. Until now, only limited efforts have been invested in utilizing GO for gene therapy in pancreatic cancer treatments. In this study, we utilized multi-functionalized monolayer GO as a gene delivery system to efficiently co-deliver HDAC1 and K-Ras siRNAs (small interfering RNAs targeting the HDAC1 gene and the G12C mutant K-Ras gene, respectively) to specifically target pancreatic cancer cells MIA PaCa-2. The systematic mechanistic elucidation of the dual gene silencing effects indicated the inactivation of both the HDAC1 and the K-Ras gene, thereby causing apoptosis, proliferation inhibition and cell cycle arrest in treated MIA PaCa-2 cells. The synergistic combination of gene silencing and NIR light thermotherapy showed significant anticancer efficacy, inhibiting in vivo tumor volume growth by >80%. Furthermore, GO can be metabolized in the mouse model within a reasonable period of time without obvious side effects. Based on preliminary in vivo application, this study for the first time indicates the promising potential of functionalized GO as a vehicle for gene therapy delivery with low toxicity for the treatment of pancreatic adenocarcinoma.

Dissecting the phenotypes of Plk1 inhibition in cancer cells using novel kinase inhibitory chemical CBB2001

Polo-like kinase 1 (Plk1) is a mitotic serine/threonine kinase and its kinase activity is closely interrelated to cell cycle progression, various types of cancer development and often correlates with poor prognosis. Thus, it is of prime importance to characterize the phenotypes of Plk1 inhibition in cells for drug development and clinical application. Here, we report a novel kinase inhibitory chemical, CBB2001, which specifically inhibited Plk1 kinase activity in vitro with an IC50 of 0.39u2009μM. In cervical carcinoma HeLa cells, we found that treatment of CBB2001 caused mitotic cell cycle arrest (EC50=0.72u2009μM) and induction of ‘polo’ cells (EC50=0.32u2009μM). Interestingly, the cell cycle arrest induced by CBB2001 was associated with accumulation of Plk1 (EC50=0.61u2009μM) and Geminin (EC50=0.43u2009μM) proteins, but distinct from the phenotypes induced by Aurora kinase inhibitors. The inhibitory effects of CBB2001 were phenocopied by RNA interferences of Plk1. We also confirmed the cell cycle inhibitory effects of CBB2001 in other cancer cells. Moreover, CBB2001 inhibited the growth of HeLa cells with an IC50 of 0.85u2009μM in MTT assays, which is better than that of reported Plk1 inhibitory chemicals ON01910 (IC50=6.46u2009μM) and LFM-A13 (IC50=37.36u2009μM). CBB2001 also inhibited mouse xenograft tumor growth. Furthermore, CBB2001 inhibited mitotic exit and delayed degradation of APC/C substrates, Geminin, Cyclin B1 and Aurora A. These specific phenotypes may serve as specific features for Plk1 inhibition and for Plk1-based clinic trials.

Black phosphorus quantum dot based novel siRNA delivery systems in human pluripotent teratoma PA-1 cells

As a novel semiconducting material, the inherent, direct, and appreciable band gap endows BP with preferable optical and electronic properties other than graphene and transition metal dichalcogenides. In addition, bio-related applications with equal importance also attract great attention thanks to several inherited advantages of BP including large drug loading capacity, high PDT efficiency, high biocompatibility and degradability. However, to date there is limited research about the biomedical applications of BP. In this study, we reported the engineering of polyelectrolyte polymers coated BP quantum dots (BP-QDs)-based nanocarriers to deliver small interfering RNA (siRNA) into human ovarian teratocarcinoma PA-1 cells. Compared to the commercial delivery reagents, superior transfection efficiency of BP-QD was detected. The expression of the LSD1 (lysine-specific demethylase 1) mRNA in PA-1 cells was significantly suppressed by BP-QDs-LSD1 siRNA complex. Notably, BP-QDs possess excellent biocompatibility and low cytotoxicity even at concentrations as high as 5 mg mL-1. The combination treatment of BP nanodots-LSD1 siRNA complex with NIR light could inhibit the cell growth rate by more than 80%. In conclusion, this is the first application of BP-QDs as gene delivery systems, which shows promising potential for siRNA delivery and photothermal effects in cancer therapy.

Structural Basis of Inhibition of ERα-Coactivator Interaction by High-Affinity N-Terminus Isoaspartic Acid Tethered Helical Peptides

Direct inhibition of the protein-protein interaction of ERα and its endogenous coactivators with a cell permeable stabilized peptide may offer a novel, promising strategy for combating ERα positive breast cancers. Here, we report the co-crystal structure of a helical peptide stabilized by a N-terminal unnatural cross-linked aspartic acid (TD) in complex with the ERα ligand binding domain (LBD). We designed a series of peptides and peptide 6 that showed direct and high-affinity binding to ERα with selective antiproliferative activity in ERα positive breast cancer cells. The co-crystal structure of the TD-stabilized peptide 6 in complex with ERα LBD further demonstrates that it forms an α helical conformation and directly binds at the coactivator binding site of ERα. Further studies showed that peptide 6W could potently inhibit cellular ERαs transcriptional activity. This approach demonstrates the potential of TD stabilized peptides to modulate various intracellular protein-protein interactions involved in a range of disorders.

TCF3 inhibits F9 embryonal carcinoma growth by the down-regulation of Oct4

T-cell factor 3 (TCF3), a downstream effector of Wnt signaling in embryonic stem (ES) cells, plays an important role in pluripotent self-renewal and proliferation. Loss of TCF3 delays the differentiation of mouse ES cells. The purpose of this study was to investigate the effect of TCF3 on embryonal carcinoma (EC). The mouse F9 EC cell line and a tumor-bearing mouse model were used to evaluate the anti-EC tumor effects of TCF3 inxa0vitro and inxa0vivo, respectively. The overexpression of TCF3 significantly inhibited proliferation, colony-forming and migration in F9 EC cells by approximately 30, 45 and 30%, respectively. The inxa0vivo mouse model showed that the overexpression of TCF3 significantly reduced tumor volume (36.4%) and tumor weight (34.8%), malignancy progression and local infiltration and prolonged the life span of tumor-bearing mice. Overexpression of TCF3 significantly down-regulated Oct4 expression in the F9 EC cells. The results indicate that TCF3 is an inhibitor of the malignant phenotypes of embryonal carcinoma through the regulation of Oct4 expression.

Development of Stabilized Peptide-Based PROTACs against Estrogen Receptor α

Peptide modulators targeting protein-protein interactions (PPIs) exhibit greater potential than small-molecule drugs in several important aspects including facile modification and relative large contact surface area. Stabilized peptides constructed by variable chemistry methods exhibit improved peptide stability and cell permeability compared to that of the linears. Herein, we designed a stabilized peptide-based proteolysis-targeting chimera (PROTAC) targeting estrogen receptor α (ERα) by tethering an N-terminal aspartic acid cross-linked stabilized peptide ERα modulator (TD-PERM) with a pentapeptide that binds the Von Hippel-Lindau (VHL) E3 ubiquitin ligase complex. The resulting heterobifunctional peptide (TD-PROTAC) selectively recruits ERα to the VHL E3 ligase complex, leading to the degradation of ERα in a proteasome-dependent manner. Compared with the control peptides, TD-PROTAC shows significantly enhanced activities in reducing the transcription of the ERα-downstream genes and inhibiting the proliferation of ERα-positive breast cancer cells. In addition, in vivo experiments indicate that TD-PROTAC leads to tumor regression in the MCF-7 mouse xenograft model. This work is a successful attempt to construct PROTACs based on cell-permeable stabilized peptides, which significantly broadens the chemical space of PROTACs and stabilized peptides.

Regulation of DNA Replication and Chromosomal Polyploidy by the MLL-WDR5-RBBP5 Methyltransferases

ABSTRACT DNA replication licensing occurs on chromatin, but how the chromatin template is regulated for replication remains mostly unclear. Here, we have analyzed the requirement of histone methyltransferases for a specific type of replication: the DNA re-replication induced by the downregulation of either Geminin, an inhibitor of replication licensing protein CDT1, or the CRL4CDT2 ubiquitin E3 ligase. We found that siRNA-mediated reduction of essential components of the MLL-WDR5-RBBP5 methyltransferase complexes including WDR5 or RBBP5, which transfer methyl groups to histone H3 at K4 (H3K4), suppressed DNA re-replication and chromosomal polyploidy. Reduction of WDR5/RBBP5 also prevented the activation of H2AX checkpoint caused by re-replication, but not by ultraviolet or X-ray irradiation; and the components of MLL complexes co-localized with the origin recognition complex (ORC) and MCM2-7 replicative helicase complexes at replication origins to control the levels of methylated H3K4. Downregulation of WDR5 or RBBP5 reduced the methylated H3K4 and suppressed the recruitment of MCM2-7 complexes onto replication origins. Our studies indicate that the MLL complexes and H3K4 methylation are required for DNA replication but not for DNA damage repair. Summary: DNA replication or re-replication of DNA induced after loss of Geminin or CLR4CDT2 is regulated by the methylation activities of the MLL-WDR5-RBBP5 methyltransferases on histone H3 at lysine 4 (H3K4).