NKp44-Derived Peptide Binds Proliferating Cell Nuclear Antigen and Mediates Tumor Cell Death.

Proliferating cell nuclear antigen (PCNA) is considered as a hub protein and is a key regulator of DNA replication, repair, cell cycle control, and apoptosis. PCNA is overexpressed in many cancer types, and PCNA overexpression is correlated with cancer virulence. Membrane-associated PCNA is a ligand for the NKp44 (NCR2) innate immune receptor. The purpose of this study was to characterize the PCNA-binding site within NKp44. We have identified NKp44-derived linear peptide (pep8), which can specifically interact with PCNA and partly block the NKp44-PCNA interaction. We then tested whether NKp44-derived pep8 (NKp44-pep8) fused to cell-penetrating peptides (CPPs) can be employed for targeting the intracellular PCNA for the purpose of anticancer therapy. Treatment of tumor cells with NKp44-pep8, fused to R11-NLS cell-penetrating peptide (R11-NLS-pep8), reduced cell viability and promoted cell death, in various murine and human cancer cell lines. Administration of R11-NLS-pep8 to tumor-bearing mice suppressed tumor growth in the 4T1 breast cancer and the B16 melanoma in vivo models. We therefore identified the NKp44 binding site to PCNA and further developed an NKp44-peptide-based agent that can inhibit tumor growth through interfering with the function of intracellular PCNA in the tumor cell.

Expression of functional alternative telomerase RNA component gene in mouse brain and in motor neurons cells protects from oxidative stress.

Telomerase, a ribonucleoprotein, is highly expressed and active in many tumor cells and types, therefore it is considered to be a target for anti-cancer agents. On the other hand, recent studies demonstrated that activation of telomerase is a potential therapeutic target for age related diseases. Telomerase mainly consists of a catalytic protein subunit with a reverse transcription activity (TERT) and an RNA component (TERC), a long non-coding RNA, which serves as a template for the re-elongation of telomeres by TERT. We previously showed that TERT is highly expressed in distinct neuronal cells of the mouse brain and its expression declined with age. To understand the role of telomerase in non-mitotic, fully differentiated cells such neurons we here examined the expression of the other component, TERC, in mouse brain. Surprisingly, by first using bioinformatics analysis, we identified an alternative TERC gene (alTERC) in the mouse genome. Using further experimental approaches we described the presence of a functional alTERC in the mouse brain and spleen, in cultures of motor neurons- like cells and neuroblastoma tumor cells. The alTERC is similar (87%) to mouse TERC (mTERC) with a deletion of 18 bp in the TERC conserved region 4 (CR4). This alTERC gene is expressed and its product interacts with the endogenous mTERT protein and with an exogenous human TERT protein (hTERT) to form an active enzyme. Overexpression of the alTERC and the mTERC genes, in mouse motor neurons like cells, increased the activity of TERT without affecting its protein level. Under oxidative stress conditions, alTERC significantly increased the survival of motor neurons cells without altering the level of TERT protein or its activity.The results suggest that the expression of the alTERC gene in the mouse brain provides an additional way for regulating telomerase activity under normal and stress conditions and confers protection to neuronal cells from oxidative stress.

Modification of topoisomerases in mammospheres derived from breast cancer cell line: clinical implications for combined treatments with tyrosine kinase inhibitors.

BACKGROUND: Accumulating evidences suggest that tumors are driven by a small population of cells, termed "cancer stem cells" (CSCs), which may be resistant to current therapeutic approaches. In breast carcinoma, the CSCs have been identified as a CD44+/CD24- cell population. These rare cells are able to grow as non-adherent sphere-like structures, termed "mammospheres", which enables their isolation and expansion in culture. To design efficient strategies for the complete eradication of CSCs, it is important to identify enzymes and proteins that are known as anti-cancer targets, and differ in their properties from those present in the none CSCs. Here we investigated the activity and expression of type I and type II DNA topoisomerases (topo I and topo II) in CSCs and their response to anti-topoisomerase inhibitors. METHODS: MCF7 breast cancer cells, PC3 prostate cancer cells and 4 T1-Luc-Oct3/4pG mouse mammary carcinoma cells were grown on low-attachment dishes in specific medium and allowed to form spheres. Enrichment of CSC population was verified by immunostaining, flow cytometry or fluorescent microscopy imaging. Nuclear protein extracts were prepared and topoisomerases activity and protein levels were determined. Cell viability was examined by the MTT and Neutral Red assays. RESULTS: Unlike the adherent MCF7 cell line, topo I activity is decreased and topo II activity is increased in the CSCs. However, the relative levels of the enzyme proteins were similar in both mammospheres and adherent cells. Topo I activity in mammospheres is regulated, at least in part, by PARP-1, as observed by the recovery of topo I activity after treatment with PARP-1 inhibitor 3-Aminobenzamide. Mammosphere-derived cells show reduced sensitivity to topo I inhibitor, camptothecin, and increased sensitivity to topo II inhibitor etoposide. Intact mammospheres show increased resistance to both drugs. A combined treatment of intact mammospheres with either CPT and gefitinib, or etoposide and erlotinib, increased the anti-cancer effect of both drugs. CONCLUSIONS: The data of this study suggest that the understanding of biological behavior of essential enzymes such as topoisomerases, in CSCs' progression and early stages of tumor development, is important for developing new strategies for cancer treatment as well as new therapies for advanced disease.

Topoisomerase I as a target of erlotinib and gefitinib: efficacy of combined treatments with camptothecin.

Topoisomerases are essential nuclear enzymes that work to resolve topological problems that normally occur during DNA metabolism. Their involvement in crucial DNA associated-processes, such as replication, transcription and repair, mark them as a target of chemotherapeutic drugs such as camptothecins (CPTs). Therefore, finding other agents that may alter their activity is of great importance. Previous data showed that certain tyrosine kinase antagonists, tyrphostins, inhibit the catalytic activity of the cellular topoisomerase I (topo I). We examined the effect of clinically used tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, on topo I in breast and prostate cancer cells. While erlotinib and gefitinib inhibit cellular topo I in treated cells without affecting the levels of the enzyme protein, in vitro assays show that erlotinib, but not gefitinib, inhibits the DNA relaxation activity of purified topo I. Erlotinib was found to reduce the DNA-binding ability of topo I, however, the reduction in topo I activity in gefitinib-treated cells is probably due to post-translational modifications of the enzyme protein. A combined treatment of either erlotinib or gefitinib with CPT increased the effect of CPT on the activity of cellular topo I, which supports the increased anticancer effect observed in MCF7 cells. These results suggest that topo I is a novel target of erlotinib and a combination of TKIs with topo I inhibitors may be an effective treatment for breast cancer.