Shao Chun Wang

Tyrosine phosphorylation controls PCNA function through protein stability.

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.

Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor.

ABSTRACT The cell membrane receptor ErbB-2 migrates to the nucleus. However, the mechanism of its nuclear translocation is unclear. Here, we report a novel mechanism of its nuclear localization that involves interaction with the transport receptor importin β1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin β1 using small interfering RNA oligonucleotides or inactivation of small GTPase Ran by RanQ69L, a dominant-negative mutant of Ran, causes a nuclear transport defect of ErbB-2. Mutation of a putative nuclear localization signal in ErbB-2 destroys its interaction with importin β1 and arrests nuclear translocation, while inactivation of nuclear export receptor piles up ErbB-2 within the nucleus. Additionally, blocking of internalization by a dominant-negative mutant of dynamin halts its nuclear localization. Thus, the cell membrane-embedded ErbB-2, through endocytosis using the endocytic vesicle as a vehicle, importin β1 as a driver and Nup358 as a traffic light, migrates from the cell surface to the nucleus. This novel mechanism explains how a receptor tyrosine kinase on the cell surface can be translocated into the nucleus. This pathway may serve as a general mechanism to allow direct communication between cell surface receptors and the nucleus, and our findings thus open a new era in understanding direct trafficking between the cell membrane and nucleus.

The ets protein PEA3 suppresses HER-2/neu overexpression and inhibits tumorigenesis

Because HER-2/neu overexpression is important in cancer development, we looked for a method of suppressing the cell transformation mediated by HER-2/neu overexpression. We have identified that the DNA-binding protein PEA3, which is encoded by a previously isolated gene of the ets family, specifically targeted a DNA sequence on the HER-2/neu promoter and downregulated the promoter activity. Expression of PEA3 resulted in preferential inhibition of cell growth and tumor development of HER-2/neu-overexpressing cancer cells. This is a new approach to targeting HER-2/neu overexpression and also provides a rationale to the design for repressors of diseases caused by overexpression of pathogenic genes.

APOBEC3G promotes liver metastasis in an orthotopic mouse model of colorectal cancer and predicts human hepatic metastasis

Colorectal cancer is the second leading cause of death from cancer in the United States. Metastases in the liver, the most common metastatic site for colorectal cancer, are found in one-third of the patients who die of colorectal cancer. Currently, the genes and molecular mechanisms that are functionally critical in modulating colorectal cancer hepatic metastasis remain unclear. Here, we report our studies using functional selection in an orthotopic mouse model of colorectal cancer to identify a set of genes that play an important role in mediating colorectal cancer liver metastasis. These genes included APOBEC3G, CD133, LIPC, and S100P. Clinically, we found these genes to be highly expressed in a cohort of human hepatic metastasis and their primary colorectal tumors, suggesting that it might be possible to use these genes to predict the likelihood of hepatic metastasis. We have further revealed what we believe to be a novel mechanism in which APOBEC3G promotes colorectal cancer hepatic metastasis through inhibition of miR-29-mediated suppression of MMP2. Together, our data elucidate key factors and mechanisms involved in colorectal cancer liver metastasis, which could be potential targets for diagnosis and treatment.

AIM2 suppresses human breast cancer cell proliferation in vitro and mammary tumor growth in a mouse model

IFN-inducible proteins are known to mediate IFN-directed antitumor effects. The human IFN-inducible protein absent in melanoma 2 (AIM2) gene encodes a 39-kDa protein, which contains a 200-amino-acid repeat as a signature of HIN-200 family (hematopoietic IFN-inducible nuclear proteins). Although AIM2 is known to inhibit fibroblast cell growth in vitro, its antitumor activity has not been shown. Here, we showed that AIM2 expression suppressed the proliferation and tumorigenicity of human breast cancer cells, and that AIM2 gene therapy inhibited mammary tumor growth in an orthotopic tumor model. We further showed that AIM2 significantly increased sub-G1 phase cell population, indicating that AIM2 could induce tumor cell apoptosis. Moreover, AIM2 expression greatly suppressed nuclear factor-κB transcriptional activity and desensitized tumor necrosis factor-α–mediated nuclear factor-κB activation. Together, these results suggest that AIM2 associates with tumor suppression activity and may serve as a potential therapeutic gene for future development of AIM2-based gene therapy for human breast cancer. [Mol Cancer Ther 2006;5(1):1–7]

DOC-2/hDab-2 inhibits ILK activity and induces anoikis in breast cancer cells through an Akt-independent pathway

DOC-2/hDab-2 was identified due to the loss of its expression in primary ovarian cancer cells. It is believed that loss of DOC-2/hDab-2 expression is one of the early events of ovarian malignancy. These results suggest a function of DOC-2/hDab-2 as a tumor suppressor. However, it is not clear how DOC-2/hDab-2 negatively regulates cancer cell growth. In this report, we demonstrate that DOC-2/hDab-2 expression in breast cancer cells resulted in sensitivity to suspension-induced cell death (anoikis). This event was associated with the down-regulation of the integrin-linked kinase (ILK) activity. Since ILK is a key factor in regulating the cellular signaling in responding to the extracellular signals through adhesion molecules like integrins, our results indicate that DOC-2/hDab-2 may prevent tumor growth and invasion by modulating the anti-apoptotic ILK pathway.

β-catenin nuclear localization is associated with grade in ovarian serous carcinoma

OBJECTIVE beta-Catenin has been previously associated with oncogenic activity in human cancers. We evaluated whether beta-catenin also plays a role in papillary serous ovarian neoplasms. METHODS Immunohistochemistry for beta-catenin was performed on the primary ovarian serous neoplasms of 105 women. Of these, 10 were low malignant potential (LMP) serous tumors, and 95 were serous cancers. Nuclear beta-catenin staining was correlated with grade of tumor and median survival. OVCAR-3, OVCA-420, OVCA-432, and MDAH-277-10c were evaluated for beta-catenin localization and transfected with a T-cell factor (TCF) responsive reporter to evaluate beta-catenin transcriptional activity. RESULTS Of 105 serous tumors, 13 (12.3%) demonstrated beta-catenin nuclear staining. Eleven of 48 high-grade serous carcinomas (23.0%) demonstrated nuclear staining compared with 1 low-grade serous carcinoma (2.1%) (P = 0.006). One LMP tumor had nuclear staining. beta-Catenin nuclear localization was undetectable in the cell lines tested. Furthermore, transient transfection of the cell lines with a TCF-responsive reporter did not demonstrate significant constitutive transcriptional activation. CONCLUSIONS We found a statistically significant correlation between beta-catenin nuclear localization and ovarian high-grade serous carcinomas. Thus, deregulation of beta-catenin may play a role in the pathogenesis of ovarian high-grade serous carcinomas in contrast to ovarian low-grade serous carcinomas and LMP serous tumors.

Upregulation of IKKα/IKKβ by integrin-linked kinase is required for HER2/neu-induced NF-κB antiapoptotic pathway

Constitutively active HER2/neu activates nuclear factor kappa-B (NF-κB) in cells and induces their resistance to apoptotic stimuli such as tumor necrosis factor-α (TNF-α). Here, we show that integrin-linked kinase (ILK), the crucial signal transducer in the integrin pathway, is involved in HER2/neu-mediated activation of NF-κB. Expression of HER2/neu increases ILK activity. Blocking ILK activity with a kinase-deficient mutant ILK (ILK-KD) inhibits NF-κB activation and sensitizes HER2/neu-transformed cells to TNF-α-induced apoptosis. Stable expression of ILK-KD in HER2/neu-transformed cells suppressed Akt phosphorylation and the expression of IκB kinase α and β (IKKα and β) at both the protein and mRNA levels, preventing IκB-α degradation and NF-κB activation. Furthermore, HER2/neu stimulated the transcriptional activity of the putative IKKβ promoter through ILK and Akt. Our results demonstrate that upregulation of IKKα and IKKβ by the ILK/Akt pathway is required for the HER2/neu-mediated NF-κB antiapoptotic pathway.

Selective Activation of Ceruloplasmin Promoter in Ovarian Tumors Potential Use for Gene Therapy

Gene therapy provides a novel treatment approach to cancer patients. Ideally, expression of therapeutic genes driven by cancer-specific promoters would only target tumors resulting in minimal toxicity to normal tissues. While there is a need of more effective and tolerable treatments for ovarian cancer patients, we aimed to identify gene promoters with high activity in ovarian tumors that can be potentially used in gene therapy to drive the expression of a therapeutic gene in tumors. To identify such promoters, a literature search was performed to reveal genes that are preferentially expressed in ovarian cancer compared with normal ovarian tissue. We found that the ceruloplasmin promoter drove up to 30-fold higher luciferase expression in ovarian cancer cells compared with immortalized normal cells. Furthermore, deletion studies revealed an activator protein-1 (AP-1) site in the ceruloplasmin promoter to be critical for optimal ceruloplasmin promoter activity. Ceruloplasmin promoter activity was significantly activated by 1-O-tetradecanoyl phorbol-13-acetate, a c-jun activator, and conversely suppressed by SP600125, a c-jun inhibitor. Consistently, the ceruloplasmin AP-1 site was specifically recognized by c-jun both in vitro and in vivo. Immunohistochemical analyses of human ovarian cancer specimens showed a direct correlation (r = 0.7, P = 0.007) between expression levels of c-jun and ceruloplasmin. In nude mice carrying SKOV3.ip1 xenografts, the ceruloplasmin promoter demonstrated significantly higher activities in tumors compared with normal organs. Together, these results suggest that the ceruloplasmin promoter activity is significantly enhanced in ovarian cancer and therefore may be exploited as a promising cancer-specific promoter in developing new gene therapy strategies for ovarian cancer.

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, by downregulating the HER‐2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and development into tumors of HER‐2/neu‐overexpressing ovarian cancer cells. Here, we establish stable clones of the human breast cancer cell line MDA‐MB‐361DYT2 that express PEA3 under the control of a tetracycline‐inducible promoter. Ectopic expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle accumulation in the G1 phase. We demonstrate that expression of PEA3 in an orthotopic breast cancer model inhibited tumor growth and prolonged the survival of tumor‐bearing mice. In a parallel experiment with another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3‐inducible transfectants, suggesting that PEA3 may exert a strong growth inhibition effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence for the antitumor activity of PEA3 in human breast cancers.