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The purpose of this phase I clinical trial was to evaluate the safety, tolerability and potential efficacy of VM202, naked DNA expressing two isoforms of hepatocyte growth factor, as an adjunct therapy to coronary artery bypass grafting (CABG) in patients with ischemic heart disease (IHD). Nine patients were assigned to receive increasing doses (0.5 to 2.0 mg) of VM202 injected into the right coronary artery (RCA) territory following completion of CABG for the left coronary artery territory. Patients were evaluated for safety and tolerability, and changes in myocardial functions were monitored via echocardiography, cardiac magnetic resonance imaging and myocardial single photon emission computed tomography throughout 6-month follow-up period. No serious complication related to VM202 was observed throughout the 6-month follow-up period. Global myocardial functions (wall motion score index, P=0.0084; stress perfusion, P=0.0002) improved during the follow-up period. In the RCA region, there was an increase in the stress perfusion (baseline vs 3-month, P=0.024; baseline vs 6-month, P=0.024) and also in the wall thickness of the diastolic and systolic phases. Intramyocardial injection of VM202 can be safely used in IHD patients with the tolerable dose of 2.0 mg. In addition, VM202 might appear to have improved regional myocardial perfusion and wall thickness in the injected region.

Ovarian endometrioid adenocarcinomas (OEAs) frequently exhibit constitutive activation of canonical WNT signaling, usually as a result of oncogenic mutations that stabilize and dysregulate the β-catenin protein. In previous work, we used microarray-based methods to compare gene expression in OEAs with and without dysregulated β-catenin as a strategy for identifying novel β-catenin/TCF target genes with important roles in ovarian cancer pathogenesis. Among the genes highlighted by the microarray studies was MSX2, which encodes a homeobox transcription factor. We found MSX2 expression was markedly increased in primary human and murine OEAs with dysregulated β-catenin compared with OEAs with intact β-catenin regulation. WNT pathway activation by WNT3a ligand or GSK3β inhibitor treatment potently induced MSX2 and ectopic expression of a dominant negative form of TCF4 inhibited MSX2 expression in ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated that β-catenin/TCF directly regulates MSX2 expression via binding to TCF binding elements in multiple regions of the MSX2 gene. Notably, ectopic MSX2 expression was found to promote neoplastic transformation of the rodent RK3E model epithelial cell line and to enhance the invasiveness of immortalized human ovarian epithelial cells in vitro and ovarian carcinoma cells in vivo. Inhibition of endogenous MSX2 expression in ovarian endometrioid cancer cells carrying a β-catenin mutation using shRNA approaches inhibited neoplastic properties of the cells in vitro and in vivo. Expression of MSX2 in selected ovarian carcinoma cells induced changes suggestive of epithelial-mesenchymal transition (EMT), but based on analysis of ovarian cell lines and primary tumor tissues, effects of MSX2 on EMT appear to be complex and context-dependent. Our findings indicate MSX2 is a direct downstream transcriptional target of β-catenin/TCF and has a key contributing role in the cancer phenotype of OEAs carrying WNT/β-catenin pathway defects.

Germline mutations of the gene encoding human fibroblast growth factor receptor 3 (FGFR3) have been shown to be responsible for several related autosomal dominant forms of syndromic craniosynostosis and short limb dwarfism. Somatic activating mutations of FGFR3 were recently reported to occur in three of 12 (25%) uterine cervical carcinomas and nine of 26 (35%) bladder carcinomas, suggesting that constitutive activation of FGFR3 may be an important mechanism underlying the development and/or progression of these common epithelial malignancies. In order to investigate further a possible role for FGFR3 mutations in cervical carcinogenesis, we performed sequence-based mutational analysis of FGFR3 in 51 primary cervical carcinomas and seven cervical carcinoma-derived cell lines. The regions analysed (exons 7, 10, 13, 15, and 19) encompassed all previously described FGFR3 mutations. A single nucleotide substitution at codon 249, predicting a serine to cysteine amino acid substitution (S249C) in the FGFR3 extracellular domain, was identified in one primary tumor. Only wild type FGFR3 alleles were identified in the remaining tumors and cell lines. The S249C mutation is the only FGFR3 mutation described to date in cervical carcinomas. These findings suggest that while activating mutations of FGFR3 occur in cervical cancer, they may not be as common as initially reported.

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.

Loss of heterozygosity and loss of expression of the deleted in colon cancer (DCC) gene is frequently observed in a number of different cancer types. To determine if the DCC gene plays a direct role in tumor suppression, wild-type full-length or truncated DCC cDNA constructs were transfected into nitrosomethylurea (NMU) transformed tumorigenic HPV-immortalized human epithelial cells that had allelic loss and reduced expression of DCC. Full-length DCC suppressed tumorigenicity whereas truncated DCC did not. Tumorigenic reversion of initially suppressed transfectants was associated with loss of DCC expression and loss or rearrangement of transfected DCC sequences. These results provide the first direct evidence that DCC is a tumor suppressor gene.

Microsatellite instability (MI), detected as electrophoretic shifts in allele sizes of microsatellite DNA sequences, has been identified in some colorectal carcinomas. Investigators have previously attributed such microsatellite instability to replication errors (RER). The colorectal carcinomas with RER have been found to arise either sporadically or in association with the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. Because endometrial carcinoma is also commonly associated with HNPCC, we studied 30 cases of endometrial carcinoma to characterize the presence of MI in these neoplasms. Seven cases (23%) showed MI. Four cases showed both Type I (large shifts) and Type II (small shifts) mutation patterns and the remaining three cases showed Type I mutations only. We conclude that MI frequently occurs in endometrial cancers and that this type of genetic alteration may be an important pathogenetic feature of this tumor type.

Allelic deletions involving chromosomes 18q occur in a significant number of colorectal cancers. Recently, a highly conserved gene called 'deleted in colorectal cancer' (DCC) has been identified on chromosome 18q. DCC has been postulated to be a colorectal tumor-suppressor gene. In order to understand the role of DCC in cell transformation, we have established a stable Rat-1 cell line expressing dexamethasone-inducible DCC antisense RNA. High levels of dexamethasone-inducible DCC antisense RNA were detected in the Rat-1 transfectants. The antisense DCC-expressing Rat-1 cells showed a faster growth rate, anchorage independence and tumorigenicity in nude mice. Exposure of the parental Rat-1 cells to antisense oligodeoxyribonucleotides to DCC resulted in inhibition of cell adhesion to the substratum which could be abrogated by various extracellular matrices. On the other hand, a bone marrow-derived stromal cell line which does not express DCC showed no detachment from the substratum when treated with the antisense oligo to DCC. These results suggest that the DCC gene is involved in cell adhesion and provide the first direct biological evidence for the possible role of DCC as a tumor-suppressor gene.