Huiling Yang

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.

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.

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.

14-3-3σ, a p53 regulator, suppresses tumor growth of nasopharyngeal carcinoma

The 14-3-3σ gene product, up-regulated by p53 in response to DNA damage, is involved in cell-cycle checkpoint control and is a human cancer epithelial marker down-regulated in various tumors. However, its role and function have not been established in nasopharyngeal carcinoma (NPC), a tumor of epithelial origin. Recently, we found that 14-3-3σ interacts with p53 in response to DNA damage and stabilizes the expression of p53. In addition, we also showed that overexpression of 14-3-3σ inhibits oncogene-activated tumorigenicity. In the present study, we investigated the tumor-suppressive role of 14-3-3σ in NPC cells. We found that there is a failure to up-regulate 14-3-3σ in response to DNA damage in two NPC cell lines that have p53 mutation. We also found that 14-3-3σ interacted with protein kinase B/Akt and negatively regulated the activity of Akt. Overexpression of 14-3-3σ inhibited NPC cell growth and blocks DNA synthesis. Overexpression of 14-3-3σ also led to inhibition of anchorage-independent growth of NPC cells. In addition, we found that 14-3-3σ sensitized NPC cells to apoptosis induced by the chemotherapeutic agent 2-methoxyestradiol. Overexpression of 14-3-3σ in both NPC cell lines reduced the tumor volume in nude mice, which could have significance for clinical application. These findings provide an insight into the roles of 14-3-3σ in NPC and suggest that approaches that modulate 14-3-3σ activity may be useful in the treatment of NPC. [Mol Cancer Ther 2006;5(2):253–60]

CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6

Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57Kip2. CSN6 associates with p57Kip2, and its overexpression can decrease the steady-state expression of p57Kip2; accordingly, CSN6 deficiency leads to p57Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57Kip2. Loss of Skp2 compromised CSN6-mediated p57Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57Kip2. CSN6’s negative impact on p57Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57Kip2 and result in promoted cancer cell growth.

Involvement of microRNA-24 and DNA methylation in resistance of nasopharyngeal carcinoma to ionizing radiation.

Nasopharyngeal carcinoma (NPC) is a malignant tumor originating in the epithelium. Radiotherapy is the standard therapy, but tumor resistance to this treatment reduces the 5-year patient survival rate dramatically. Studies are urgently needed to elucidate the mechanism of NPC radioresistance. Epigenetics—particularly microRNAs (miRNA) and DNA methylation—plays an important role in carcinogenesis and oncotherapy. We used qRT-PCR analysis and identified an miRNA signature from differentially expressed miRNAs. Our objectives were to identify the role of miR24 in NPC tumorigenesis and radioresistance and to identify the mechanisms by which miR24 is regulated. We found that miR24 inhibited NPC cell growth, promoted cell apoptosis, and suppressed the growth of NPC xenografts. We showed that miR24 was significantly downregulated in recurrent NPC tissues. When combined with irradiation, miR24 acted as a radiosensitizer in NPC cells. One of the miR24 precursors was embedded in a CpG island. Aberrant DNA methylation was involved in NPC response to radiotherapy, which linked inactivation of miR24 through hypermethylation of its precursor promoter with NPC radioresistance. Treating NPC cells with the DNA-hypomethylating agent 5-aza-2′-deoxycytidine compensated for the reduced miR24 expression. Together, our findings showed that miR24 was negatively regulated by hypermethylation of its precursor promoter in NPC radioresistance. Our findings defined a central role for miR24 as a tumor-suppressive miRNA in NPC and suggested its use in novel strategies for treatment of this cancer. Mol Cancer Ther; 13(12); 3163–74. ©2014 AACR.

Emerging roles of Jab1/CSN5 in DNA damage response, DNA repair, and cancer

Jab1/CSN5 is a multifunctional protein that plays an important role in integrin signaling, cell proliferation, apoptosis, and the regulation of genomic instability and DNA repair. Dysregulation of Jab1/CSN5 activity has been shown to contribute to oncogenesis by functionally inactivating several key negative regulatory proteins and tumor suppressors. In this review, we discuss our current understanding of the relationship between Jab1/CSN5 and DNA damage and summarize recent findings regarding opportunities for and challenges to therapeutic intervention.

Tumor suppressor ARF inhibits HER-2/ neu -mediated oncogenic growth

HER2/neu, a receptor tyrosine kinase oncogene, promotes mitogenic growth and antiapoptotic activity in cancer cells. Strong expression of HER2/neu in cancers has been associated with poor prognosis. Alternative reading frame protein (ARF), a tumor suppressor protein encoded by a gene located in the Ink4a/ARF gene locus, is frequently inactivated in human cancers. Little is known about the tumor suppressor role of ARF in HER2/neu-overexpressing cancers. Here, we applied the ARF gene as a tumor-suppressive agent for HER2/neu-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. We found that ARF antagonized protein kinase B (PKB)/Akt-mediated p27Kip1 phosphorylation and increased p27 stability in HER2/neu-overexpressing cells. ARF expression also led to decreased levels of Cul1 and Skp2, two proteins involved in p27 degradation. We also found that ARF caused apoptosis in HER2/neu-overexpressing cells, and sensitized cells to apoptosis induced by the chemotherapeutic agents taxol and 2-methoxyestradiol. Most significantly for clinical application, we found that ARF inhibited HER2/neu-mediated cell growth, transformation, and tumorigenesis. These findings indicate that modulation of ARF activity may be a useful therapeutic intervention in HER2-overexpressing cancers.

HER2-Akt signaling in regulating COP9 signalsome subunit 6 and p53

HER2/neu oncogene is frequently overexpressed in various types of cancer, and the (PI3K)-Akt signaling pathway is often activated in HER2-overexpressing cancer cells. CSN6, subunit 6 of the COP9 signalosome complex, is pivotal in regulating MDM2 to destabilize p53, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to CSN6 regulation, and that Akt is a positive regulator of CSN6. Ectopic expression of Akt can increase the expression of CSN6; accordingly, Akt inhibition leads to CSN6 destabilization. Mechanistic studies show that Akt causes CSN6 phosphorylation at Ser 60, which, in turn, reduces ubiquitin-mediated protein degradation of CSN6. Significantly, Akt’s positive impact on CSN6 elevation translates into p53 degradation, potentiating transformational activity and increasing DNA damage. Akt inhibition can attenuate these defects caused by CSN6. These data suggest that Akt is an important positive regulator of CSN6, and that activation of Akt in many types of cancer could lead to abnormal elevation of CSN6 and result in downregulated p53 and increased DNA damage, which promotes cancer cell growth.

Tumor-suppression function of transcription factor USF2 in prostate carcinogenesis

Although the transcription factor USF2 has been implicated in the regulation of cellular growth and proliferation, it is unknown whether alterations in USF2 contribute to tumorigenesis and tumor development. We examined the role of USF2 in prostate tumorigenesis. Western blot analysis revealed markedly decreased USF2 levels in three androgen-independent prostate cancer cell lines, PC-3, DU145, and M12, as compared to nontumorigenic prostate epithelial cells or the androgen-dependent cell line, LNCaP. Ectopic expression of USF2 in PC-3 cells did not affect the cell proliferation rate of PC-3 cells on plastic surfaces. However, it dramatically decreased anchorage-independent growth of PC-3 cells in soft agar (90–98% inhibition) and the invasion capability (80% inhibition) of PC-3 cells in matrix gel assay. Importantly, expression of USF2 in PC-3 cells inhibited the tumorigenicity of PC-3 cells in an in vivo nude mice xenograft model (80–90% inhibition). These results suggest that USF2 has tumor-suppression function. Consistent with its function in tumor suppression, we found that the USF2 protein is present in normal prostate epithelial cells but absent in 18 of 42 (43%) human prostate cancer tissues (P=0.015). To further examine the functional role of USF2 in vivo, we generated mice with genetic deletion of USF2 gene. We found that USF2-null mice displayed marked prostate hyperplasia at a young age, suggesting that USF2 is involved in the normal growth and differentiation of prostate. Together, these studies demonstrate that USF2 has tumor-suppressor function and plays a role in prostate carcinogenesis.