The increasing complexity of the oncofetal h19 gene locus: functional dissection and therapeutic intervention.

The field of the long non-coding RNA (lncRNA) is advancing rapidly. Currently, it is one of the most popular fields in the biological and medical sciences. It is becoming increasingly obvious that the majority of the human transcriptome has little or no-protein coding capacity. Historically, H19 was the first imprinted non-coding RNA (ncRNA) transcript identified, and the H19/IGF2 locus has served as a paradigm for the study of genomic imprinting since its discovery. In recent years, we have extensively investigated the expression of the H19 gene in a number of human cancers and explored the role of H19 RNA in tumor development. Here, we discuss recently published data from our group and others that provide further support for a central role of H19 RNA in the process of tumorigenesis. Furthermore, we focus on major transcriptional modulators of the H19 gene and discuss them in the context of the tumor-promoting activity of the H19 RNA. Based on the pivotal role of the H19 gene in human cancers, we have developed a DNA-based therapeutic approach for the treatment of cancers that have upregulated levels of H19 expression. This approach uses a diphtheria toxin A (DTA) protein expressed under the regulation of the H19 promoter to treat tumors with significant expression of H19 RNA. In this review, we discuss the treatment of four cancer indications in human subjects using this approach, which is currently under development. This represents perhaps one of the very few examples of an existing DNA-based therapy centered on an lncRNA system. Apart from cancer, H19 expression has been reported also in other conditions, syndromes and diseases, where deregulated imprinting at the H19 locus was obvious in some cases and will be summarized below. Moreover, the H19 locus proved to be much more complicated than initially thought. It houses a genomic sequence that can transcribe, yielding various transcriptional outputs, both in sense and antisense directions. The major transcriptional outputs of the H19 locus are presented here.

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.

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

BACKGROUND: 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. METHODS: The therapeutic potential of the toxin vectors driven by the H19 and the IGF2-P3 regulatory sequences was tested in a metastatic model of rat CC531 colon carcinoma in liver. RESULTS: Intratumoral injection of these vectors into colon tumors implanted in the liver of rats induced an 88% and a 50% decrease respectively in the median tumor volume as compared with the control groups. This therapeutic action was accompanied by increased necrosis of the tumor. Importantly, no signs of toxicity were detected in healthy animals after their treatment by the toxin expression vectors. CONCLUSIONS: DT-A was preferentially expressed in liver metastases after being transfected with H19 or IGF2-P3 promoter-driven DT-A expression plasmids, causing a very significant inhibition of tumor growth as a result of its cytotoxic effect. Our findings strongly support the feasibility of our proposed therapeutic strategy, which may contribute to open new gene therapeutic options for human liver metastases.

Gene expression in the bladder carcinoma rat model.

We investigated gene expression in N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced rat bladder carcinoma in order to test its applicability as a model for the study of novel therapeutic modalities, particularly gene therapy. We administered BBN in the drinking water to Wistar rats for up to 30 wk and induced papillary transitional cell carcinoma (TCC), which is similar to the most prevalent type of human bladder cancer. Tumor evolution was similar to that found in previous studies. However, we described the morphological stages according to modern human bladder carcinoma terminology. Our main goal was to examine the expression levels of the H19 gene, of the insulin-like growth factor 2 (Igf2) transcripts expressed from promoters P2 and P3 and of the telomerase subunits that we had previously investigated as tools for targeted gene therapy of bladder cancer. We detected at 30 wk of BBN exposure significant upregulation of these sequences in the rat bladder tumors, similar to our previous findings in human bladder cancer. To reinforce the similarity of this model to the corresponding human disease, we searched for additional tumor-specific genes documented as having altered expression in human bladder carcinoma, using cDNA expression arrays (Clontech). We suggest that BBN-induced rat bladder cancer has morphological, biological, and molecular parallels to human bladder cancer and is an attractive model for studying novel alternatives of therapeutic intervention.

Oncofetal splice-pattern of the human H19 gene.

H19 is an imprinted gene that demonstrates maternal monoallelic expression in fetal tissues and in some cancers, and very likely does not code for a protein. H19 is involved in the regulation of cell proliferation, embryonic growth, and differentiation through upstream and downstream cis elements that influence the expression of IGF2, a closely physically linked gene, and also through its RNA involved in metastasis and angiogenic processes. We report the identification of an alternatively spliced variant of H19 RNA that lacks part of exon 1. This variant was detected in human embryonic and placental tissues, but not in bladder or hepatocellular carcinomas. A very low level of this variant was also detected in colon carcinoma. The observed pattern of expression suggests that this splice variant is a developmentally regulated H19 gene transcript.

Possible physiological role of H19 RNA.

The product of the imprinted oncofetal H19 gene is an untranslated RNA of unknown function. With the human cDNA Atlas microarray, we detected differentially expressed genes modulated by the presence of H19 RNA. Many of the genes that are upregulated by H19 RNA are known to contribute to the invasive, migratory, and angiogenic capacities of cells. Moreover, we provided experimental data indicating that whereas H19 RNA did not have any growth advantage for the cells when cultured in 10% fetal calf serum, it did confer an advantage when cells were cultured in serum-poor medium. This observation can be explained in part by the inability of the H19-expressing cells to induce the cyclin-dependent kinase inhibitor p57(kip2) in response to serum stress. Our results favor the possible role of the H19 gene in promoting cancer progression, angiogenesis, and metastasis.

Use of H19 regulatory sequences for targeted gene therapy in cancer.

We present a tumor gene therapy approach based on the use of regulatory sequences of the H19 gene that are differentially expressed between normal and cancer cells. We constructed expression vectors carrying the gene for the A fragment of diphtheria toxin (DT-A) or herpes simplex virus thymidine kinase (HSV-tk), under the control of a 814 bp 5'-flanking region of the H19 gene. The cell killing activity of these constructs was in accordance with the relative activity of the H19 regulatory sequences in the transfected cells. We evaluated the therapeutic potential of the gene expression constructs driven by H19 regulatory sequences in an animal model of bladder cancer induced by subcutaneous injection of syngeneic bladder tumor cell lines. Intratumoral injection of these constructs caused a significant suppression of subcutaneous tumor growth, with no obvious toxicity toward the host.

Analysis of differentially expressed genes in hepatocellular carcinoma using cDNA arrays.

Hepatocellular carcinoma (HCC) is characterized by multiple somatic mutations, including DNA rearrangements, that affect many cell-growth regulatory pathways. Many genes differentially expressed in HCC have been reported previously, but the patterns of expression varied significantly between patients who bore different risk factors for HCC. To identify genes whose differential expression could serve as a "signature" for diagnosis and prognosis of HCC, we performed analyses of differentially expressed genes in three cases of HCC with different risk factors using the Atlas Human Cancer cDNA Expression Arrays. Among all 597 genes present on the array, only three were found to be coordinately differentially expressed in all three HCC cases, in agreement with published data. These three genes, Cu/Zn superoxide dismutase, osteonectin/secreted protein acidic and rich in cysteine, and matrix metalloproteinase 14, could serve as candidates for the HCC "signature." Ten genes were found to be coordinately differentially expressed in only two of three tested HCC cases. On the other hand, many genes that had been reported previously as differentially expressed in HCC failed to show the described pattern of expression in this group. The results of this study confirm the great variability in gene-expression patterns in HCC and establish the utility of the array technology for identifying both the HCC signature genes and individual gene-expression patterns for purposes of patient-oriented therapy.