Junjian Wang

Kinesin family deregulation coordinated by bromodomain protein ANCCA and histone methyltransferase MLL for breast cancer cell growth, survival, and tamoxifen resistance.

Kinesins are a superfamily of motor proteins and often deregulated in different cancers. However, the mechanism of their deregulation has been poorly understood. Through examining kinesin gene family expression in estrogen receptor (ER)-positive breast cancer cells, we found that estrogen stimulation of cancer cell proliferation involves a concerted regulation of specific kinesins. Estrogen strongly induces expression of 19 kinesin genes such as Kif4A/4B, Kif5A/5B, Kif10, Kif11, Kif15, Kif18A/18B, Kif20A/20B, Kif21, Kif23, Kif24, Kif25, and KifC1, whereas suppresses the expression of seven others, including Kif1A, Kif1C, Kif7, and KifC3. Interestingly, the bromodomain protein ANCCA/ATAD2, previously shown to be an estrogen-induced chromatin regulator, plays a crucial role in the up- and downregulation of kinesins by estrogen. Its overexpression drives estrogen-independent upregulation of specific kinesins. Mechanistically, ANCCA (AAA nuclear coregulator cancer associated) mediates E2-dependent recruitment of E2F and MLL1 histone methyltransferase at kinesin gene promoters for gene activation–associated H3K4me3 methylation. Importantly, elevated levels of Kif4A, Kif15, Kif20A, and Kif23 correlate with that of ANCCA in the tumors and with poor relapse-free survival of patients with ER-positive breast cancer. Their knockdown strongly impeded proliferation and induced apoptosis of both tamoxifen-sensitive and resistant cancer cells. Together, the study reveals ANCCA as a key mediator of kinesin family deregulation in breast cancer and the crucial role of multiple kinesins in growth and survival of the tumor cells. Implications: These findings support the development of novel inhibitors of cancer-associated kinesins and their regulator ANCCA for effective treatment of cancers including tamoxifen-resistant breast cancers. Mol Cancer Res; 12(4); 539–49. ©2014 AACR.

ROR-[gamma] drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer

The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoic acid receptor–related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits nuclear receptor coactivator 1 and 3 (NCOA1 and NCOA3, also known as SRC-1 and SRC-3) to an AR–ROR response element (RORE) to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of both AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR target gene network. Finally, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing, but not AR-negative, xenograft PCa models, and they effectively sensitized CRPC tumors to enzalutamide, without overt toxicity, in mice. Taken together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and as a potential therapeutic target for advanced PCa.

Reprogramming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes

Metabolic reprogramming such as the aerobic glycolysis or Warburg effect is well recognized as a common feature of tumorigenesis. However, molecular mechanisms underlying metabolic alterations for tumor therapeutic resistance are poorly understood. Through gene expression profiling analysis we found that histone H3K36 methyltransferase NSD2/MMSET/WHSC1 expression was highly elevated in tamoxifen-resistant breast cancer cell lines and clinical tumors. IHC analysis indicated that NSD2 protein overexpression was associated with the disease recurrence and poor survival. Ectopic expression of NSD2 wild type, but not the methylase-defective mutant, drove endocrine resistance in multiple cell models and xenograft tumors. Mechanistically, NSD2 was recruited to and methylated H3K36me2 at the promoters of key glucose metabolic enzyme genes. Its overexpression coordinately up-regulated hexokinase 2 (HK2) and glucose-6-phosphate dehydrogenase (G6PD), two key enzymes of glycolysis and the pentose phosphate pathway (PPP), as well as TP53-induced glycolysis regulatory phosphatase TIGAR. Consequently, NSD2-driven tamoxifen-resistant cells and tumors displayed heightened PPP activity, elevated NADPH production, and reduced ROS level, without significantly altered glycolysis. These results illustrate a coordinated, epigenetic activation of key glucose metabolic enzymes in therapeutic resistance and nominate methyltransferase NSD2 as a potential therapeutic target for endocrine resistant breast cancer.

Estrogen-related receptor alpha confers methotrexate resistance via attenuation of reactive oxygen species production and P53 mediated apoptosis in osteosarcoma cells.

Osteosarcoma (OS) is a malignant tumor mainly occurring in children and adolescents. Methotrexate (MTX), a chemotherapy agent, is widely used in treating OS. However, treatment failures are common due to acquired chemoresistance, for which the underlying molecular mechanisms are still unclear. In this study, we report that overexpression of estrogen-related receptor alpha (ERRα), an orphan nuclear receptor, promoted cell survival and blocked MTX-induced cell death in U2OS cells. We showed that MTX induced ROS production in MTX-sensitive U2OS cells while ERRα effectively blocked the ROS production and ROS associated cell apoptosis. Our further studies demonstrated that ERRα suppressed ROS induction of tumor suppressor P53 and its target genes NOXA and XAF1 which are mediators of P53-dependent apoptosis. In conclusion, this study demonstrated that ERRα plays an important role in the development of MTX resistance through blocking MTX-induced ROS production and attenuating the activation of p53 mediated apoptosis signaling pathway, and points to ERRα as a novel target for improving osteosarcoma therapy.

Curcumin Promotes Osteosarcoma Cell Death by Activating miR-125a/ERRα Signal Pathway.

Curcumin has demonstrated valuable therapeutic potential against a variety of human cancers including osteosarcoma. However, the molecular mechanisms underlying its anti‐tumor effect remain to be poorly understood. By RNA sequence profiling, we found that curcumin significantly down‐regulates the expression of estrogen‐related receptor alpha (ERRα) in osteosarcoma cells. Overexpression of ERRα diminished curcumin‐activated apoptotic cell death and scavenged curcumin‐induced reactive oxygen species (ROS), while ERRα silencing sensitized osteosarcoma cells to curcumin, resulting in increased inhibition of cell proliferation. In addition, we found that curcumin suppressed the ERRα gene expression through upregulation of miR‐125a. Data from this study revealed a novel mechanism for curcumin‐mediated apoptotic cell death, which involves tumor cell killing via activating miR‐125a/ERRα pathway. Our studies also provide further support for osteosarcoma therapy by targeting ERRα alone or in combination with curcumin. J. Cell. Biochem. 118: 74–81, 2017.

Silencing the epigenetic silencer KDM4A for TRAIL and DR5 simultaneous induction and antitumor therapy.

Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, has a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound-4 (C-4) or gene silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at promoters of TRAIL and DR5 transcriptional activator CHOP gene by dissociating KDM4A and nuclear receptor corepressor (NCoR)-HDAC complex and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics.

Erratum: ROR-γ 3 drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer (Nature Medicine (2016))

Nat. Med.; doi:10.1038/nm.4070; corrected online 22 April 2016 In Figure 2a of the version of this article initially published online, one phenyl ring was inadvertently deleted from the chemical structure of compound SR2211. One affiliation of H.-W.C. (Veterans Affairs Northern California Health Care System–Mather, Mather, California, USA) was also inadvertently omitted.

Transporting Cells in Semi-Solid Gel Condition and at Ambient Temperature.

Mammalian cells including human cancer cells are usually transported in cryovials on dry ice or in a liquid nitrogen vapor shipping vessel between different places at long distance. The hazardous nature of dry ice and liquid nitrogen, and the associated high shipping cost strongly limit their routine use. In this study, we tested the viability and properties of cells after being preserved or shipped over long distance in Matrigel mixture for different days. Our results showed that cells mixed with Matrigel at suitable ratios maintained excellent viability (>90%) for one week at room temperature and preserved the properties such as morphology, drug sensitivity and metabolism well, which was comparable to cells cryopreserved in liquid nitrogen. We also sent cells in the Matrigel mixture via FedEx service to different places at ambient temperature. Upon arrival, it was found that over 90% of the cells were viable and grew well after replating. These data collectively suggested that our Matrigel-based method was highly convenient for shipping live cells for long distances in semi-solid gel condition and at ambient temperature.

Structure-Based Discovery and Optimization of Benzo[ d]isoxazole Derivatives as Potent and Selective BET Inhibitors for Potential Treatment of Castration-Resistant Prostate Cancer (CRPC)

The bromodomain and extra-terminal (BET) family proteins have gained increasing interest as drug targets for treatment of castration-resistant prostate cancer (CRPC). Here, we describe the design, optimization, and evaluation of benzo[ d]isoxazole-containing compounds as potent BET bromodomain inhibitors. Cocrystal structures of the representative inhibitors in complex with BRD4(1) provided solid structural basis for compound optimization. The two most potent compounds, 6i (Y06036) and 7m (Y06137), bound to the BRD4(1) bromodomain with Kd values of 82 and 81 nM, respectively. They also exhibited high selectivity over other non-BET subfamily members. The compounds potently inhibited cell growth, colony formation, and the expression of AR, AR regulated genes, and MYC in prostate cancer cell lines. Compounds 6i and 7m also demonstrated therapeutic effects in a C4-2B CRPC xenograft tumor model in mice. These potent and selective BET inhibitors represent a new class of compounds for the development of potential therapeutics against CRPC.

Benzoxazinone-containing 3,5-dimethylisoxazole derivatives as BET bromodomain inhibitors for treatment of castration-resistant prostate cancer

The bromodomain and extra-terminal proteins (BET) have emerged as promising therapeutic targets for the treatment of castration-resistant prostate cancer (CRPC). We report the design, synthesis and evaluation of a new series of benzoxazinone-containing 3,5-dimethylisoxazole derivatives as selective BET inhibitors. One of the new compounds, (R)-12 (Y02234), binds to BRD4(1) with a Kd value of 110 nM and blocks bromodomain and acetyl lysine interactions with an IC50 value of 100 nM. It also exhibits selectivity for BET over non-BET bromodomain proteins and demonstrates reasonable anti-proliferation and colony formation inhibition effect in prostate cancer cell lines such as 22Rv1 and C4-2B. The BRD4 inhibitor (R)-12 also significantly suppresses the expression of ERG, Myc and AR target gene PSA at the mRNA level in prostate cancer cells. Treatment with (R)-12 significantly suppresses the tumor growth of prostate cancer (TGI = 70%) in a 22Rv1-derived xenograft model. These data suggest that compound (R)-12 is a promising lead compound for the development of a new class of therapeutics for the treatment of CRPC.