DNA vaccine elicits an efficient antitumor response by targeting the mutant Kras in a transgenic mouse lung cancer model.

Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.

GATA3 targets semaphorin 3B in mammary epithelial cells to suppress breast cancer progression and metastasis.

Semaphorin 3B (SEMA3B) is a secreted axonal guidance molecule that is expressed during development and throughout adulthood. Recently, SEMA3B has emerged as a tumor suppressor in non-neuronal cells. Here, we show that SEMA3B is a direct target of GATA3 transcriptional activity. GATA3 is a key transcription factor that regulates genes involved in mammary luminal cell differentiation and tumor suppression. We show that GATA3 relies on SEMA3B for suppression of tumor growth. Loss of SEMA3B renders GATA3 inactive and promotes aggressive breast cancer development. Overexpression of SEMA3B in cells lacking GATA3 induces a GATA3-like phenotype and higher levels of SEMA3B are associated with better cancer patient prognosis. Moreover, SEMA3B interferes with activation of LIM kinases (LIMK1 and LIMK2) to abrogate breast cancer progression. Our data provide new insights into the role of SEMA3B in mammary gland and provides a new branch of GATA3 signaling that is pivotal for inhibition of breast cancer progression and metastasis.

Antagonism of p53-dependent apoptosis by mitogen signals.

p53-mediated apoptosis is antagonized by growth factor stimulation. Here, we show that p53-dependent cell death induced by DNA damage was effectively prevented by mitogen activation. The levels of Bcl-2, Bcl-xL, and Bax were not altered by cisplatin treatment and mitogen rescue. Instead, the protection against p53-regulated apoptosis was mediated by at least three distinct signaling pathways. Either phosphatidylinositol (PI) 3-kinase or mitogen-activated protein kinase kinase (MEK) antagonized p53-induced apoptosis, and an additive preventive effect was observed when both kinases were activated. However, the combination of PI 3-kinase and MEK was not sufficient to completely prevent apoptosis induced by DNA damage. Mitogen activation further suppressed cisplatin-induced p53 expression, and the inhibition was mainly dependent on the Ca2+ pathway. Our results demonstrate that effective antagonism of p53-dependent apoptosis by mitogenic activation requires the presence of multiple signal pathways, including PI 3-kinase, MEK, and Ca2+.

Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells.

Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other's expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial-mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.

Induction of antitumor immunity with combination of HER2/neu DNA vaccine and interleukin 2 gene-modified tumor vaccine.

The therapeutic effects of both cytokine-secreting tumor vaccine and DNA vaccine were studied using mouse MBT-2 bladder cancer cells as a model. Cytokine-secreting MBT-2 cells were obtained by infecting cells with retroviral particles containing interleukin (IL) 2-, IL-4-, or granulocyte-macrophage colony-stimulating factor (GM-CSF)-expression vector. The MBT-2-IL-2 cells were not tumorigenic in syngenic C3H mice at all. Tumor formation decreased significantly for the MBT-2-GM-CSF cells. MBT-2-IL-2, -IL-4, and -GM-CSF cells were killed by irradiation and tested as tumor vaccines. The irradiated MBT2-IL-2 cells could complete protect mice from the growth of the preexisting tumor cells, and the immune memory lasted for 8 months. On the other hand, irradiated MBT-2-IL-4 and MBT-2-GM-CSF cells were less effective. When the loading tumor mass increased, all tumor vaccines lost protective effects. DNA vaccine encoding the tumor antigen neu was additionally tested to improve the therapeutic efficacy. Coinjection of 60 microg pSV-neu DNA was effective in enhancing the antitumor effects of MBT2-IL-2; however, DNA vaccine alone cannot prevent the progression of the preexisting tumor. Immunohistochemical analysis of tumor infiltrate revealed massive increase of CD4+ lymphoid cells in the group of mice treated with both DNA vaccine and IL-2-secreted tumor vaccine. Western blotting demonstrated the presence of anti-neu antibody in the serum from immunized mice. In contrast, combination of DNA vaccine and MBT-2-GM-CSF has no additive effect. The results indicate the combination of DNA vaccine and IL-2-secreting tumor vaccine can additionally improve therapeutic efficacy, and the efficacy is correlated with the increase of CD4+ T lymphocytes and anti-neu antibody.

Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese.

Cytochrome P450 (CYP) 2C9 catalyses the metabolism of a wide range of drugs. Previous studies have shown the differences in the amino acid composition among CYP2C9 variants at Cys144/Arg, Tyr358/Cys, Leu359/Ile, and Gly417/Asp. PCR-endonuclease digestion methods have been developed to detect these four possible polymorphisms. The T416-->C mutation in exon 3 of CYP2C9 (Cys144-->Arg) creates an Ava II site. In the 135 subjects we tested, all leukocyte DNA samples showed a complete Ava II digestion indicating homozygous C416 (Arg144). A Tyr358-->Cys mutation will create a Nsi I site at codon 1057-1063 in exon 7. In 40 subjects tested, all samples showed negative results. DNA sequencing on a few samples showed Tyr358Ile359. A mismatched PCR primer pair was then designed to detect codon C1061-->A (Leu359-->Ile) mutation. In 115 subjects tested, 111 samples showed a complete Nsi I digestion (Ile359) and four samples showed heterozygous results. Another mismatched PCR primer pair was used to confirm the C1061 codon in heterozygous subjects. The four heterozygous subjects showed partial digestion with endonuclease Kpn I, which confirmed the heterozygous Ile/Leu at amino acid 359. The G1236-->A mutation in exon 8 of CYP2C9 (Gly417-->Asp) creates a Hph I site. In all 46 subjects, homozygous G1236 (Gly417) was found. Most Chinese subjects actually have Arg144 Tyr358 Ile359 Gly417 in CYP2C9 as previously reported human-2. Furthermore, we found an A-->T (+12 position in intron 2) mutation in our CYP2C9 sequencing process. The mutation creates a NIa III site.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of morphine from codeine in Chinese subjects of different CYP2D6 genotypes.

Codeine and morphine pharmacokinetics among different CYP2D6 genotypes was compared in this study. Polymerase chain reaction tests were used to determine CYP2D6 genotypes in leukocyte deoxyribonucleic acid in 32 unrelated volunteers. Based on the genotypes, subjects were categorized into three groups: homozygous C/C188 (n = 8), heterozygous C/T188 (n = 12), and homozygous T/T188 (n = 12). Each subject was given a single oral dose of 30 mg codeine phosphate tablet after overnight fasting. Plasma concentration of codeine and 24-hour urinary morphine recovery were measured with HPLC. All three genotypes of subjects showed almost identical time profiles of plasma codeine. Urinary morphine glucuronide was hydrolyzed with beta-glucuronidase. The total recovered amount of morphine and glucuronides was 4349 +/- 646, 2564 +/- 242, and 1127 +/- 164 nmol (mean +/- SEM), respectively, for C/C188, C/T188, and T/T188 subjects (p < 0.05). The significant lower amount of urinary morphine but identical codeine plasma concentration suggested a lower partial clearance of the formation of morphine from codeine in T/T188 subjects. The results suggest a future study to assess the analgesic effect of codeine in different genotypes of CYP2D6 extensive metabolizers.