Ruiying Zhao

MiR-205 determines the radioresistance of human nasopharyngeal carcinoma by directly targeting PTEN.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), but radioresistance severely reduces NPC radiocurability. Here, we have established a radio-resistant NPC cell line, CNE-2R, and investigate the role of miRNAs in radioresistance. The miRNAs microarray assay reveals that miRNAs are differentially expressed between CNE-2R and its parental cell line CNE-2. We find that miR-205 is elevated in CNE-2R. A target prediction algorithm suggests that miR‑205 regulates expression of PTE N, a tumor-suppressor. Introducing miR-205 into CNE-2 cells suppresses PTE N protein expression, followed by activation of AKT, increased number of foci formation and reduction of cell apoptosis postirradiation. On the other hand, knocking down miR-205 in CNE-2R cells compromises the inhibition of PTE N and increases cell apoptosis. Significantly, immunohistochemistry studies demonstrate that PTE N is downregulated at late stages of NPC, and that miR-205 is significantly elevated followed the radiotherapy. Our data conclude that miR-205 contributes to radioresistance of NPC by directly targeting PTE N. Both miR-205 and PTE N are potential predictive biomarkers for radiosensitivity of NPC and may serve as targets for achieve successful radiotherapy in NPC.

Roles of COP9 signalosome in cancer

The constitutive photomorphogenesis 9 signalosome (COP9 or CSN) is an evolutionarily conserved multiprotein complex found in plants and animals. Because of the homology between the COP9 signalosome and the 19S lid complex of the proteosome, COP9 has been postulated to play a role in regulating the degradation of polyubiquitinated proteins. Many tumor suppressor and oncogene products are regulated by ubiquitination- and proteosome-mediated protein degradation. Therefore, it is conceivable that COP9 plays a significant role in cancer, regulating processes relevant to carcinogenesis and cancer progression (e.g., cell cycle control, signal transduction and apoptosis). In mammalian cells, it consists of eight subunits (CSN1 to CSN8). The relevance and importance of some subunits of COP9 to cancer are emerging. However, the mechanistic regulation of each subunit in cancer remains unclear. Among the CSN subunits, CSN5 and CSN6 are the only two that each contain an MPN (Mpr1p and Pad1p N-terminal) domain. The deneddylation activity of an MPN domain toward cullin-RING ubiquitin ligases (CRL) may coordinate CRL-mediated ubiquitination activity. More recent evidence shows that CSN5 and CSN6 are implicated in ubiquitin-mediated proteolysis of important mediators in carcinogenesis and cancer progression. Here, we discuss the mechanisms by which some CSN subunits are involved in cancer to provide a much needed perspective regarding COP9 in cancer research, hoping that these insights will lay the groundwork for cancer intervention.

DNA Damage–Induced Protein 14-3-3 σ Inhibits Protein Kinase B/Akt Activation and Suppresses Akt-Activated Cancer

14-3-3 sigma is induced by tumor suppressor protein p53 in response to DNA damage. p53 can directly transactivate the expression of 14-3-3 sigma to cause a G(2) cell cycle arrest when cell DNA is damaged. The expression of 14-3-3 sigma protein is down-regulated in various tumors, but its function has not been fully established. Protein kinase B/Akt, a crucial regulator of oncogenic signal involved in cell survival and proliferation, is deregulated in many types of cancer. Akt activation can enhance p53 degradation, but its role in DNA damage response is not clear. Here, we show that Akt activation is diminished when p53 and 14-3-3 sigma is up-regulated in response to DNA damage. Evidence is provided that 14-3-3 sigma binds and inhibits Akt. In keeping with this concept, Akt-mediated cell survival is inhibited by 14-3-3 sigma. Significantly, we show that 14-3-3 sigma inhibits Akt-mediated cell growth, transformation, and tumorigenesis. Low expression of 14-3-3 sigma in human primary breast cancers correlates with Akt activation. These data provide an insight into Akt regulation and rational cancer gene therapy by identifying 14-3-3 sigma as a molecular regulator of Akt and as a potential anticancer agent for Akt-activated cancers.

Pim-1 plays a pivotal role in hypoxia-induced chemoresistance

Hypoxia changes the responses of cancer cells to many chemotherapy agents, resulting in chemoresistance. The underlying molecular mechanism of hypoxia-induced drug resistance remains unclear. Pim-1 is a survival kinase, which phosphorylates Bad at serine 112 to antagonize drug-induced apoptosis. Here we show that hypoxia increases Pim-1 in a hypoxia-inducible factor-1α-independent manner. Inhibition of Pim-1 function by dominant-negative Pim-1 dramatically restores the drug sensitivity to apoptosis induced by chemotherapy under hypoxic conditions in both in vitro and in vivo tumor models. Introduction of siRNAs for Pim-1 also resensitizes cancer cells to chemotherapy drugs under hypoxic conditions, whereas forced overexpression of Pim-1 endows solid tumor cells with resistance to cisplatin, even under normoxia. Dominant-negative Pim-1 prevents a decrease in mitochondrial transmembrane potential in solid tumor cells, which is normally induced by cisplatin (CDDP), followed by the reduced activity of Caspase-3 and Caspase-9, indicating that Pim-1 participates in hypoxia-induced drug resistance through the stabilization of mitochondrial transmembrane potential. Our results demonstrate that Pim-1 is a pivotal regulator involved in hypoxia-induced chemoresistance. Targeting Pim-1 may improve the chemotherapeutic strategy for solid tumors.

Constitutively active FOXO4 inhibits Akt activity, regulates p27 Kip1 stability, and suppresses HER2-mediated tumorigenicity

The FOXO family of Forkhead transcription factors, regulated by the phosphoinositide-3-kinase–Akt pathway, is involved in cell cycle regulation and apoptosis. Strong expression of HER2, a receptor tyrosine kinase oncogene, in cancers has been associated with a poor prognosis. Recently, FOXO4 was shown to regulate the transcription of the cyclin-dependent kinase inhibitor p27 Kip1 gene directly. Also, we have shown that HER2 promotes mitogenic growth and transformation of cancer cells by downregulation of p27 Kip1. Given the fact that FOXO4 mediates p27 transcription, we hypothesize that an Akt phosphorylation mutant of FOXO4 (FOXO4A3), which maintains the activity to transactivate p27 Kip1, may be used as an anticancer agent for HER2-overexpressing cancers. Here, we applied the FOXO4 gene as a novel anticancer agent for HER2-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. Overexpression of FOXO4A3 inhibits HER2-activated cell growth. We found that FOXO4A3 inhibited the kinase activity of protein kinase B/Akt and reversed HER2-mediated p27 mislocation in the cytoplasm. FOXO4A3 expression also led to decreased levels of CSN5, a protein involved in p27 degradation. These data suggest that FOXO4A3 also can regulate p27 post-transcriptionally. In addition, we found that FOXO4A3 sensitized cells to apoptosis induced by the chemotherapeutic agent 2-methoxyestradiol. Most significantly for clinical application, FOXO4A3 expression in HER2-overexpressing cells can be regulated in vivo and reduces the tumor volume in a tumor model. These findings indicate the applicability of employing FOXO4 regulation as a therapeutic intervention in HER2-overexpressing cancers.

Modeling Familial Cancer with Induced Pluripotent Stem Cells

In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs.

Hypoxia-Mediated Up-Regulation of Pim-1 Contributes to Solid Tumor Formation

Tumor hypoxia directly promotes genomic instability and facilitates cell survival, resulting in tumors with a more aggressive phenotype. The proto-oncogene pim-1 regulates apoptosis and the cell cycle by phosphorylating target proteins. Overexpression of Pim-1 can cause genomic instability and contribute to lymphomagenesis. It is not clear whether Pim-1 is involved in hypoxia-mediated tumor survival in solid tumors. Here, we show that hypoxia can stabilize Pim-1 by preventing its ubiquitin-mediated proteasomal degradation and can cause Pim-1 translocation from the cytoplasm to the nucleus. Importantly, overexpression of Pim-1 increases NIH3T3 cell transformation exclusively under hypoxic conditions, suggesting that Pim-1 expression under hypoxia may be implicated in the transformation process of solid tumors. Also, blocking Pim-1 function by introduction of dominant negative Pim-1 resensitizes pancreatic cancer cells to apoptosis induced by glucose-deprivation under hypoxia. Introduction of short interfering RNAs for Pim-1 also resensitizes cancer cells to glucose deprivation under hypoxic conditions, while forced overexpression of Pim-1 causes solid tumor cells to become resistant to glucose deprivation. Moreover, dominant negative Pim-1 reduces tumorigenicity in pancreatic cancer cells and HeLa xenograft mouse models. Together, our studies indicate that Pim-1 plays a distinct role in solid tumor formation in vivo, implying that Pim-1 may be a novel target for cancer therapy.

Subunit 6 of the COP9 signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers

The mammalian constitutive photomorphogenesis 9 (COP9) signalosome (CSN), a protein complex involved in embryonic development, is implicated in cell cycle regulation and the DNA damage response. Its role in tumor development, however, remains unclear. Here, we have shown that the COP9 subunit 6 (CSN6) gene is amplified in human breast cancer specimens, and the CSN6 protein is upregulated in human breast and thyroid tumors. CSN6 expression positively correlated with expression of murine double minute 2 (MDM2), a potent negative regulator of the p53 tumor suppressor. Expression of CSN6 appeared to prevent MDM2 autoubiquitination at lysine 364, resulting in stabilization of MDM2 and degradation of p53. Mice in which Csn6 was deleted died early in embryogenesis (E7.5). Embryos lacking both Csn6 and p53 survived to later in embryonic development (E10.5), which suggests that loss of p53 could partially rescue the effect of loss of Csn6. Mice heterozygous for Csn6 were sensitized to γ-irradiation-induced, p53-dependent apoptosis in both the thymus and the developing CNS. These mice were also less susceptible than wild-type mice to γ-irradiation-induced tumorigenesis. These results suggest that loss of CSN6 enhances p53-mediated tumor suppression in vivo and that CSN6 plays an important role in regulating DNA damage-associated apoptosis and tumorigenesis through control of the MDM2-p53 signaling pathway.

Effects of Obesity on Transcriptomic Changes and Cancer Hallmarks in Estrogen Receptor–Positive Breast Cancer

Background Obesity increases the risk of cancer death among postmenopausal women with estrogen receptor–positive (ER+) breast cancer, but the direct evidence for the mechanisms is lacking. The purpose of this study is to demonstrate direct evidence for the mechanisms mediating this epidemiologic phenomenon. Methods We analyzed transcriptomic profiles of pretreatment biopsies from a prospective cohort of 137 ER+ breast cancer patients. We generated transgenic (MMTV-TGFα;A y /a) and orthotopic/syngeneic (A y /a) obese mouse models to investigate the effect of obesity on tumorigenesis and tumor progression and to determine biological mechanisms using whole-genome transcriptome microarrays and protein analyses. We used a coculture system to examine the impact of adipocytes/adipokines on breast cancer cell proliferation. All statistical tests were two-sided. Results Functional transcriptomic analysis of patients revealed the association of obesity with 59 biological functional changes (P < .05) linked to cancer hallmarks. Gene enrichment analysis revealed enrichment of AKT-target genes (P = .04) and epithelial–mesenchymal transition genes (P = .03) in patients. Our obese mouse models demonstrated activation of the AKT/mTOR pathway in obesity-accelerated mammary tumor growth (3.7- to 7.0-fold; P < .001; n = 6–7 mice per group). Metformin or everolimus can suppress obesity-induced secretion of adipokines and breast tumor formation and growth (0.5-fold, P = .04; 0.3-fold, P < .001, respectively; n = 6–8 mice per group). The coculture model revealed that adipocyte-secreted adipokines (eg, TIMP-1) regulate adipocyte-induced breast cancer cell proliferation and invasion. Metformin suppress adipocyte-induced cell proliferation and adipocyte-secreted adipokines in vitro. Conclusions Adipokine secretion and AKT/mTOR activation play important roles in obesity-accelerated breast cancer aggressiveness in addition to hyperinsulinemia, estrogen signaling, and inflammation. Metformin and everolimus have potential for therapeutic interventions of ER+ breast cancer patients with obesity.

Negative cell cycle regulator 14-3-3σ stabilizes p27 Kip1 by inhibiting the activity of PKB/Akt

The 14-3-3σ (sigma) protein is a human cancer marker downregulated in various tumors, but its function has not been fully established. 14-3-3σ is a negative regulator of cell cycle when overexpressed, but it is not clear whether 14-3-3σ regulates cyclin-dependent kinase inhibitor p27Kip1 to negatively affect cell cycle progression. Protein kinase B/Akt is a crucial regulator of oncogenic signal and can phosphorylate p27Kip1 to enhance p27Kip1degradation, thereby promoting cell growth. Here, we show that 14-3-3σ-mediated cell cycle arrest concurred with p27Kip1 upregulation and Akt inactivation. We show that 14-3-3σ blocks Akt-mediated acceleration of p27Kip1 turnover rate. 14-3-3σ inhibits Akt-mediated p27Kip1 phosphorylation that targets p27Kip1 for nuclear export and degradation. 14-3-3σ inhibits cell survival and tumorigenicity of Akt-activating breast cancer cell. Low expression of 14-3-3σ in human primary breast cancers correlates with cytoplasmic location of p27Kip1. These data provide an insight into 14-3-3σ activity and rational cancer gene therapy by identifying 14-3-3σ as a positive regulator of p27 and as a potential anticancer agent.