Oncofetal H19 RNA promotes tumor metastasis.

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-ß and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-ß treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.

Development of targeted therapy for ovarian cancer mediated by a plasmid expressing diphtheria toxin under the control of H19 regulatory sequences.

BACKGROUND: Ovarian cancer ascites fluid (OCAF), contains malignant cells, is usually present in women with an advanced stage disease and currently has no effective therapy. Hence, we developed a new therapy strategy to target the expression of diphtheria toxin gene under the control of H19 regulatory sequences in ovarian tumor cells. H19 RNA is present at high levels in human cancer tissues (including ovarian cancer), while existing at a nearly undetectable level in the surrounding normal tissue. METHODS: H19 gene expression was tested in cells from OCAF by the in-situ hybridization technique (ISH) using an H19 RNA probe. The therapeutic potential of the toxin vector DTA-H19 was tested in ovarian carcinoma cell lines and in a heterotopic animal model for ovarian cancer. RESULTS: H19 RNA was detected in 90% of patients with OCAF as determined by ISH. Intratumoral injection of DTA-H19 into ectopically developed tumors caused 40% inhibition of tumor growth. CONCLUSION: These observations may be the first step towards a major breakthrough in the treatment of human OCAF, while the effect in solid tumors required further investigation. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure.

H19-promoter-targeted therapy combined with gemcitabine in the treatment of pancreatic cancer.

Pancreatic cancer is the eighth cancer leading cause of cancer-related death in the world and has a 5-year survival rate of 1-4% only. Gemcitabine is a first line agent for advanced pancreatic therapy; however, its efficacy is limited by its poor intracellular metabolism and chemoresistance. Studies have been conducted in an effort to improve gemcitabine treatment results by adding other chemotherapeutic agents, but none of them showed any significant advantage over gemcitabine monotherapy. We found that 85% of human pancreatic tumors analyzed by in situ hybridization analyses showed moderated to strong expression of the H19 gene. We designed a preclinical study combining gemcitabine treatment and a DNA-based therapy for pancreatic cancer using a non viral vector BC-819 (also known as DTA-H19), expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The experiments conducted either in an orthotopic and heterotopic pancreatic carcinoma animal model showed better antitumor activity following the sequential administration of the vector BC-819 and gemcitabine as compared to the effect of each of them alone. The results presented in the current study indicate that treatment with BC-819 in combination with gemcitabine might be a viable new therapeutic option for patients with advanced pancreatic cancer.

Targeting diphtheria toxin and TNF alpha expression in ovarian tumors using the H19 regulatory sequences.

BACKGROUND: There are currently no effective therapies for the treatment of ovarian cancer ascites fluid (OCAF). H19 is an RNA oncofetal gene that is present at high levels in human cancer tissues (ovarian cancer and OCAF among them), while existing at a nearly undetectable level in the surrounding normal tissue. There is evidence for a synergistic effect in cell cytotoxicity mediated by TNFα and diphtheria toxin in sensitive and resistant human ovarian tumor cell line. Thus, we tested the cytotoxic effect of TNF-α cytokine, together with the diphtheria toxin, in the therapy of ovarian cancer. METHODS: The therapeutic potential of toxin vectors carrying the DT-A gene alone (pH19-DTA), or in combination with the TNF-α gene (pH19-TNF-DTA), driven by H19 regulatory sequences were tested in ovarian carcinoma cell lines and in a heterotopic ovarian cancer model. RESULTS: The toxin vectors showed a high killing capacity when transfected into different ovarian cancer cell lines. In addition, intratumoral injection of the toxin vector into ectopically developed tumors caused 40% inhibition of tumor growth. The killing effect after injection of pH19-TNF-DTA plasmid into ectopically developed tumors was significantly higher than that showed by the pH19-DTA plasmid alone, particularly in diphtheria toxin and TNF resistant tumors. CONCLUSIONS: These observations may be the first step towards a major breakthrough in the treatment of human ovarian cancer. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure coupled with unnecessary suffering and cost.

Treatment of ovarian cancer ascites by intra-peritoneal injection of diphtheria toxin A chain-H19 vector: a case report.

INTRODUCTION: Ovarian cancer ascitic fluid, which contains malignant cells, is usually present in women with an advanced stage disease. There are currently no effective therapies for the treatment of ovarian cancer ascitic fluid. We developed a new therapeutic strategy to target expression of the diphtheria toxin fragment A gene in ovarian tumor cells under the control of H19 regulatory sequences. CASE PRESENTATION: A 64-year-old Caucasian woman was diagnosed with a stage IIIc epithelial ovarian cancer. She suffered from progressive disease, accumulation of malignant ascites that needed to be drained weekly, abdominal pain, vomiting, anorexia and severe weakness. Infusion of the diphtheria toxin A chain-H19 plasmid into the peritoneum of our patient resulted in complete resolution of the ascites with minimum adverse events. CONCLUSION: On the basis of this preliminary experience, we are currently conducting an extensive Phase I study on a larger number of patients in order to assess the safety and preliminary efficacy of this novel patient-oriented treatment approach.