Aya Mizrahi

The oncofetal H19 RNA connection: Hypoxia, p53 and cancer

Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53(wt)) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53(null) cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-alpha as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-alpha abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53(null) hypoxic cells. In p53(wt) hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-alpha are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-alpha activity. In vivo a significant increase in H19 expression occurred in tumors derived from p53(null) cells but not in p53(wt) cells. Taken together, our results indicate that a functional link exists between p53, HIF1-alpha and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.

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

BackgroundOvarian 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.MethodsH19 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.ResultsH19 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.ConclusionThese 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.

Regulatory sequences of H19 and IGF2 genes in DNA-based therapy of colorectal rat liver metastases

Malignant tumors of the liver are among the most common causes of cancer‐related death throughout the world. Current therapeutic approaches fail to control the disease in most cases. This study seeks to explore the potential utility of transcriptional regulatory sequences of the H19 and insulin growth factor 2 (IGF2) genes for directing tumor‐selective expression of a toxin gene (A fragment of diphtheria toxin), delivered by non‐viral vectors.

Regional therapy with DTA-H19 vector suppresses growth of colon adenocarcinoma metastases in the rat liver.

Curative surgery is possible in barely 10% of patients with colorectal liver metastases and combined treatment modalities scarcely improve survival in this group of patients. Hence, investigations of new therapeutic modalities are crucial. Overexpression of the H19 gene in liver metastases points to H19 as a target for cancer gene therapy. Here we have evaluated the possibility of regional intra-arterial treatment of liver meta-stases with the DTA-H19 plasmid. Intra-arterial treatment of a total dose of 2.5 mg (repeated injections of 500 µg DTA-H19 plasmid each dose after the first injection of 1000 µg) caused a significant delay in the tumor growth compared to control group. All of the tumors treated with the control vector increased in size, whereas 35.7% of the tumors in the groups treated with a total amount of 2.5 mg DTA-H19 plasmid shrank in size. The present study showed that the DTA-H19 plasmid administered intra-arterially significantly delayed the tumor growth and even resulted in tumor regression in high percentage of the treated animals with liver metastases of colon cancer. Since human liver metastases demonstrated overexpression of the H19 gene, regional administration of the plasmid seems to be a promising therapeutic approach.

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

IntroductionOvarian 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 presentationA 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.ConclusionOn 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.

30. Patient Oriented DNA Based Therapy from the Bench to the Patient

The objective of the present study is to develop novel DNA based therapy strategies for bladder cancer. The successful development of anti-tumor gene therapy depends on the use of a combinatorial approach aimed at targeted delivery and specific expression of effective anti-tumor agents. Our laboratory characterized in depth the H19 expression profile in tumors. We seek to explore the potential of regulatory sequences of the H19 gene and of other differentially expressed genes in tumors for directing tumor-selective expression of toxins or suicide genes delivered by non-viral vectors. In this tumor-specific approach of DNA based therapy we use the differences in the expression profiles of a tumor compared to that of its surrounding normal tissue in an individual patient to tailor the most efficient therapy. We propose to use the regulatory sequences of the imprinted gene H19 for the development of DNA based therapy of human bladder carcinoma.