Chaehwa Park

Interleukin 12 Gene Therapy of Cancer by Peritumoral Injection of Transduced Autologous Fibroblasts: Outcome of a Phase I Study

A phase I dose-escalation clinical trial of peritumoral injections of interleukin 12 (IL-12)-transduced autologous fibroblasts was performed in patients with disseminated cancer for whom effective treatment does not exist. The goals of this study were to assess the safety and toxicities as well as the efficacy, and ancillarily the immunomodulatory effects, of peritumoral IL-12 gene transfer. Primary dermal fibroblasts cultured from the patients were transduced with retroviral vector carrying human IL-12 genes (p35 and p40) as well as the neomycin phosphotransferase gene (TFG-hIL-12-Neo). Patients received four injections at intervals of 7 days. Nine patients were enrolled in this dose-escalation study, with secreted IL-12 doses ranging from 300 ng/24 hr for the first three patients to 1000, 3000, and 5000 ng/24 hr for two patients in each subsequent dosage level. Although a definite statement cannot be made, there appears to be perturbation of systemic immunity. Also, the locoregional effects mediated by tumor necrosis factor alpha (TNF-alpha) and CD8+ T cells were observed with tumor regression. Treatment-related adverse events were limited to mild to moderate pain at the injection site; clinically significant toxicities were not encountered. Transient but clear reductions of tumor sizes were observed at the injected sites in four of nine cases, and at noninjected distant sites in one melanoma patient. Hemorrhagic necrosis of tumors was observed in two melanoma patients. These data indicate that gene therapy by peritumoral injection of IL-12-producing autologous fibroblasts is feasible, and promising in patients with advanced cancer.

Effect of Simvastatin on Cetuximab Resistance in Human Colorectal Cancer With KRAS Mutations

BACKGROUND Metastatic colorectal cancer (CRC) patients with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are resistant to treatment with cetuximab, a monoclonal antibody that targets the epidermal growth factor receptor. Statins have reported antitumor activity, but it is unknown whether simvastatin can reverse cetuximab resistance in KRAS mutant CRC. METHODS Human CRC cell lines with KRAS mutations (LS153, LS174T, DLD1, LoVo, SW403, SW480, SNU175, and LS1034) or with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations (DiFi, SW48, HT29, and RKO) were used to test the effect of cetuximab, simvastatin, and cetuximab plus simvastatin on cell proliferation and apoptosis in vitro. Because BRAF(V600E) mutant may be responsible for cetuximab resistance in KRAS wild-type cells, we measured the growth of xenograft tumors originating from KRAS mutant and BRAF mutant cells in mice treated with cetuximab alone or plus simvastatin (n = 5 mice per treatment group). We used immunoblot assays to study RAS-regulated activation of BRAF protein after simvastatin treatment. All statistical tests were two-sided. RESULTS Addition of simvastatin (0.2 μM) to cetuximab (0.03-1.0 μM) reduced cell proliferation of KRAS mutant (P < .001) but not of BRAF mutant CRC cells in vitro. Treatment of KRAS mutant cells with simvastatin reduced BRAF activity and induced apoptosis. Treatment with cetuximab and simvastatin reduced the growth of xenograft tumors originating from KRAS mutant cells compared with cetuximab alone (eg, for tumors originating from DLD1 cells, cetuximab vs cetuximab + simvastatin, mean tumor volume = 49.4 vs 20.2 cm(3), mean difference = 29.2 cm(3), 95% confidence interval = 19.7 to 38.5, P < .001); treatment with cetuximab alone or in combination with simvastatin had no effect on the growth of BRAF mutant tumors. CONCLUSION Simvastatin may overcome cetuximab resistance in colon cancer cells with KRAS mutations by modulating BRAF activity and inducing apoptosis.

Maspin expression is transactivated by p63 and is critical for the modulation of lung cancer progression

Maspin inhibits metastasis of some cancer cells, and clinical studies have identified correlations between maspin loss and poor prognosis in several cancer types. Maspin was found to be significantly overexpressed in lung cancer samples as compared with matched normal lung tissues. However, the regulatory mechanism of maspin expression remains unclear. We show here that differential expression of maspin in carcinoma-derived lung cancer cells is regulated at the transcriptional level. We found that p63 is a critical factor for the transcription of maspin, which is lost in highly invasive cancer cells such as NCI-H157, NCI-322, and NCI-358. No correlation was found between maspin expression and the previously associated transcription factors, p53, Ets1, and Pdef. Instead, maspin expression was strictly dependent on the presence of p63 in lung cancer tissues (P < 0.001) and in the tested cell lines. Transient expression of p63 transactivated the maspin promoter with remarkable fold changes in cells expressing the TAp63, suggesting that TAp63 might be a novel stimulator of the maspin promoter in lung cancer. We have also demonstrated the binding of p63 protein to a previously identified p53-binding site on the maspin promoter by gel shift and chromatin immunoprecipitation assays. In tumor tissues, maspin expression was associated with lymph node involvement (P = 0.035) and tumor stage (P = 0.063) in all tested cases, except squamous carcinoma. In terms of function, ectopic expression of maspin inhibited cell invasion in squamous carcinoma as well as adenocarcinoma. Taken together, these results define maspin as a new molecular target of p63 that eventually inhibits the invasion of lung cancer.

Epstein–Barr virus latent membrane protein-1 protects B-cell lymphoma from rituximab-induced apoptosis through miR-155-mediated Akt activation and up-regulation of Mcl-1

Abstract Rituximab is a CD20-targeted monoclonal antibody widely used in the treatment of B-cell lymphoma. Previously, we have shown that Epstein–Barr virus (EBV) latent membrane protein-1 (LMP1) increases chemoresistance in malignant cancer cells. In this study we examined the effects of LMP1 on the response of B-cell lymphoma cell lines to rituximab. Here we show for the first time that LMP1 activates the Akt pathway and up-regulates Mcl-1 through miR-155 expression, which contributes to the survival of rituximab-treated B-cell lymphoma cells. Furthermore, Akt inhibition or knockdown of Mcl-1 and miR-155 was found to be an efficient strategy to overcome rituximab resistance caused by LMP1 expression. Thus, we propose Akt and Mcl-1 and miR-155 as molecular targets for therapeutic intervention in the treatment of EBV-associated B-cell lymphoma with rituximab.

Maspin suppresses survival of lung cancer cells through modulation of Akt pathway.

PURPOSE Maspin is a tumor suppressor protein that has been reported to stimulate the cell death of cancer and inhibit the metastasis of cancer. The present study aimed to explore the survival pathway by which maspin modulates the resistance of human lung cancer cells to chemotherapeutic drugs, and the consequences of maspin gene therapy in an animal model. MATERIALS AND METHODS NCI-H157 and A549 cells were transfected with either a mock vector (pCMVTaq4C), maspin (pCMV-maspin), siControl or siMaspin. RT-PCR and Western blot analysis were performed to study the expressions of survival proteins in lung cancer. cDNA microarray analysis was carried out to compare the maspin-modulated gene expression between the xenograft tumors derived from the lung cancer cells that were stably transfected with pCMVTaq4C or pCMV-maspin. Maspin gene therapy was performed by intra-tumoral injections of pCMVTaq4C or pCMV-maspin into the pre-established subcutaneous tumors in nude mice. RESULTS Maspin significantly decreased the survival to doxorubicin and etoposide, whereas did not affect the survival to cisplatin in the NCI-H157 cells. Interestingly, transfection with a maspin plasmid resulted in a significant reduction of the phosphorylation of Akt in the NCI-H157 cells, whereas knockdown of maspin increased the phosphorylation of Akt in the A549 cells. Microarray analysis of the xenograft tumors revealed a specific gene expression profile, demonstrating that maspin is associated with the differential expressions of PTEN and IGF2R. Direct transfer of pCMV-maspin into the tumor significantly retarded the tumor growth in the animal experiments (p=0.0048). CONCLUSION Lung cancer cells lacking maspin could be resistant to chemotherapeutic drugs such as doxorubicin or etoposide, at least in part by maintaining Akt phosphorylation.

Epstein-Barr virus latent membrane protein 1 increases genomic instability through Egr-1-mediated up-regulation of activation-induced cytidine deaminase in B-cell lymphoma.

Abstract Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) is a transmembrane protein essential for EBV-induced immortalization and transformation of B cells. Activation-induced cytidine deaminase (AID) triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Here, we report an intracellular mechanism by which LMP1 contributes to B cell lymphomagenesis via AID expression. In our experiments, LMP1 increased AID mRNA expression and promoter activity. The AID promoter region contains a binding site for Egr-1, a prominent transcription factor that is reported to be up-regulated by LMP1. In promoter activity analysis, Egr-1 enhanced the reporter activity of the wild-type AID promoter, but not that containing a mutated Egr-1 binding site. Egr-1 knockdown abrogated LMP-1-mediated up-regulation of AID promoter reporter activity in EBV-negative BJAB cells and reduced AID promoter reporter activity in EBV-positive SKW6.4 cells. AID induced down-regulation of the nuclear factor-κB (NFκB) inhibitory tumor suppressor Rassf6, suggesting that AID functions as an upstream regulator of the NFκB inhibitory Rassf6. Moreover, Egr-1 expression was associated with an increased number of genomic lesions in genome-wide analysis using single nucleotide polymorphism (SNP) microarray and copy number variation (CNV). Collectively, LMP1 induces AID up-regulation and genomic instability via Egr-1. Increased AID expression may, in turn, promote down-regulation of the NFκB inhibitor, Rassf6, thereby further increasing the survival of genetically destabilized B-cell lymphoma cells.

A Novel Senescence-Evasion Mechanism Involving Grap2 and Cyclin D Interacting Protein Inactivation by Ras Associated with Diabetes in Cancer Cells under Doxorubicin Treatment

Ras associated with diabetes (Rad) is a Ras-related GTPase that promotes cell growth by accelerating cell cycle transitions. Rad knockdown induced cell cycle arrest and premature senescence without additional cellular stress in multiple cancer cell lines, indicating that Rad expression might be critical for the cell cycle in these cells. To investigate the precise function of Rad in this process, we used human Rad as bait in a yeast two-hybrid screening system and sought Rad-interacting proteins. We identified the Grap2 and cyclin D interacting protein (GCIP)/DIP1/CCNDBP1/HHM, a cell cycle-inhibitory molecule, as a binding partner of Rad. Further analyses revealed that Rad binds directly to GCIP in vitro and coimmunoprecipitates with GCIP from cell lysates. Rad translocates GCIP from the nucleus to the cytoplasm, thereby inhibiting the tumor suppressor activity of GCIP, which occurs in the nucleus. Furthermore, in the presence of Rad, GCIP loses its ability to reduce retinoblastoma phosphorylation and inhibit cyclin D1 activity. The function of Rad in transformation is also evidenced by increased telomerase activity and colony formation according to Rad expression level. In vivo tumorigenesis analyses revealed that tumors derived from Rad knockdown cells were significantly smaller than those from control cells (P = 0.0131) and the preestablished tumors are reduced in size after the injection of siRad (P = 0.0064). Therefore, we propose for the first time that Rad may promote carcinogenesis at least in part by inhibiting GCIP-mediated tumor suppression.

SNARK, a novel downstream molecule of EBV latent membrane protein 1, is associated with resistance to cancer cell death

To investigate the effect of Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) on human cancer cells, we sought to identify and analyse potential target genes that were differentially expressed in the presence and absence of LMP1. LMP1 upregulated the expression of SNARK compared with the empty vector transfected control cells, which was confirmed by reverse-transcription polymerase chain reaction. Cytotoxicity assay showed that SNARK expression increased drug resistance in response to doxorubicin (P = 0.0009), whereas knockdown of SNARK by siRNA (siSNARK) effectively inhibited LMP-1-mediated increase of cell survival (P = 0.0131). The expression of anti-apoptotic genes such as BCL6 and BIRC2 was increased by SNARK, and knockdown of these genes decreased SNARK-mediated increase of cell survival. The associations of SNARK, BCL6, and BIRC2 and the presence of EBV were also observed in T-cell lymphoma cell lines, NKL and HANK-1. These results suggest that SNARK is a downstream cellular target of LMP1 in malignant cells and can be a novel candidate for therapeutic intervention of EBV-associated cancer.

Influence of small interfering RNA corresponding to ets homologous factor on senescence-associated modulation of prostate carcinogenesis

Senescence is thought to be an inherent tumor-suppressive mechanism. In the process of identifying senescence-associated genes, we found significant suppression of the ets homologous factor (EHF) in cancer cells in a state of DNA damage–induced senescence. In this study, we show that EHF provides substantial drug resistance in PC-3 prostate cancer cells by inhibiting senescence and cell cycle arrest. Knockdown of EHF by small interfering RNA inhibited cell proliferation and induced a premature cellular senescence characterized by hypophosphorylation of Rb and increased level of p27, with concomitant decreases of cyclin A, cdc2, and E2F1. Telomeric repeat amplification protocol analysis showed that transient EHF knockdown significantly decreased telomerase activity, whereas this activity was increased by overexpression of EHF. In vivo tumorigenesis analyses revealed that tumors derived from EHF knockdown cells were significantly smaller than those derived from control cells (P < 0.0001). Further, the preestablished tumors were reduced after the injection of small interfering RNA corresponding to EHF (P = 0.0122). Collectively, these observations indicate that aberrant expression of EHF and the subsequent disruption of p27-mediated senescence and telomerase activity is likely to contribute significantly to tumor progression, and furthermore that EHF might be a promising target for future cancer therapeutics. [Mol Cancer Ther 2006;5(12):3191–6]

Effects of transforming growth factor β (TGF-β) receptor on lung carcinogenesis

Transforming growth factor beta (TGF-beta) type-II receptor mutations have been reported in several epithelial-type human malignancies. To elucidate the role of TGF-beta RII in lung cancer progression, we prepared gene-modified clones of the human lung cancer cell line NCI-H23. NCI-H23, a human non-small-cell lung adenocarcinoma cell line which has a frameshift mutation in, and reduced expression of, the TGF-beta type-II receptor (TGF-beta RII), exhibits resistance to growth inhibition by TGF-beta(1) in vitro. Transfection of NCI-H23 with a retroviral vector expressing wild-type TGF-beta RII restored the responsiveness of cells to exogenous TGF-beta(1) with reduced cell proliferation. Immunocytochemical analysis demonstrated nuclear translocation of Smad3 after TGF-beta(1) treatment in RII-restored NCI-H23 cells. Underphosphorylation of the retinoblastoma protein accompanying p21 up-regulation was observed after TGF-beta(1) treatment of NCI-H23-RII cells. Receptor restoration also changed the levels of VEGF mRNA induced by TGF-beta(1). However, impairment of TGF-beta signalling did not alter microvessel formation in vivo in transplanted tumours. Instead, in vivo tumorigenesis experiments revealed a remarkable difference in the number and sizes of the tumours derived from NCI-H23-RII cells and dominant negative NCI-H23-dnRII cells (P < 0.01). Collectively, these observations suggest that impairment of TGF-beta signal transduction contributes significantly to tumour progression, mainly by cell proliferation rather than by modulation of angiogenesis in human NCI-H23 lung carcinoma cells.