Tamar Schneider

Parental imprinting of the human H19 gene

It has only recently become clear that genetic imprinting plays an important role in human embryogenesis and in processes leading to the development of pediatric cancers and other human diseases. Using a unique human tissue, the androgenetic complete hydatidiform mole, we established that the maternally inherited allele of the imprinted H19 gene is expressed. Our results also show that the paternal allele of the human IGF‐II gene, a gene suspected to be parentally imprinted in humans, is expressed.

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-β 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-β 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.

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 p57kip2 in response to serum stress. Our results favor the possible role of the H19 gene in promoting cancer progression, angiogenesis, and metastasis.

The product of the imprinted H19 gene is an oncofetal RNA.

AIMS/BACKGROUND: The H19 gene is an imprinted, maternally expressed gene in humans. It is tightly linked and coregulated with the imprinted, paternally expressed gene of insulin-like growth factor 2. The H19 gene product is not translated into protein and functions as an RNA molecule. Although its role has been investigated for more than a decade, its biological function is still not understood fully. H19 is abundantly expressed in many tissues from early stages of embryogenesis through fetal life, and is down regulated postnatally. It is also expressed in certain childhood and adult tumours. This study was designed to screen the expression of H19 in human cancer and its relation to the expression of H19 in the fetus. METHODS: Using in situ hybridisation with a [35S] labelled probe, H19 mRNA was detected in paraffin wax sections of fetal tissues from the first and second trimesters of pregnancy and of a large array of human adult and childhood tumours arising from these tissues. RESULTS: The H19 gene is expressed in tumours arising from tissues which express this gene in fetal life. Its expression in the fetus and in cancer is closely linked with tissue differentiation. CONCLUSIONS: Based on these and previous data, H19 is neither a tumour suppressor gene nor an oncogene. Its product is an oncofetal RNA. The potential use of this RNA as a tumour marker should be evaluated.

Imprinted H19 oncofetal RNA is a candidate tumour marker for hepatocellular carcinoma.

AIMS/BACKGROUND: To study the expression of the H19 gene in hepatocellular carcinoma. H19 is an imprinted, maternally expressed gene, which is tightly linked, both physically and functionally, to the paternally expressed insulin-like growth factor 2 (IGF II). IGF II is known to be involved in liver carcinogenesis. H19 was first discovered in the fetal mouse liver to be under the same regulatory genes as alpha fetoprotein (alpha FP), a widely used tumour marker for hepatocellular carcinoma. METHODS: Using both radioactive and non-radioactive in situ hybridisation, the expression of the H19 gene was compared with the presence of alpha FP, as demonstrated by immunohistochemistry, in 18 cases of hepatocellular carcinoma. RESULTS: H19 expression was present in 13 of 18 cases, whereas staining for alpha FP was positive in only nine of 18 cases. Concordance was found in 12 of 18 tumours (66.7%). In general, the staining pattern for H19 was more diffuse than the immunohistochemical staining for alpha FP. CONCLUSIONS: The addition of a non-radioactive in situ hybridisation assay for H19 RNA to the panel of tumour markers used for the histopathological and cytological diagnosis of hepatocellular carcinoma might be useful.

The imprinted H19 gene as a tumor marker in bladder carcinoma

OBJECTIVES Genomic imprinting is a newly discovered mechanism in genetics that is involved in tumorigenesis. H19 is an imprinted gene in the human, expressed from the maternal allele. It is extensively transcribed in fetal life but is not translated and functions as an RNA molecule. It has been suggested as a candidate tumor suppressor gene. We studied the expression of H19 in human cancer arising from tissues expressing H19 in fetal life, one of which is bladder mucosa. METHODS In situ hybridization for H19 mRNA on paraffin sections of bladder carcinoma in different histologic grades. RESULTS Low-grade (grade 1 of 3), noninvasive (Ta) papillary transitional cell bladder carcinoma did not express H19, but prominent expression was disclosed in higher grades, invasive transitional cell carcinomas (T1-T3/4). Expression was also evident in in situ bladder carcinoma (Tis), which tends to progress rapidly to invasive cancer. CONCLUSIONS We suggest that H19 can be used as a tumor marker in human bladder carcinoma, where its expression indicates a more malignant potential.

H19 expression in hepatic metastases from a range of human carcinomas

Aims: To investigate the expression of the imprinted oncofetal H19 gene in hepatic metastases derived from a range of human carcinomas and assess its prognostic value with the view of developing a DNA based treatment for such metastases. Methods: Non-radioactive in situ hybridisation for H19 RNA was performed on paraffin wax embedded sections of liver biopsies or partial hepatectomy specimens, taken from 80 patients with hepatic metastases derived from carcinomas from several medical centres in Israel. The degree of expression was graded qualitatively according to the number of cells expressing H19 and the intensity of staining. The medical files were searched for demographic data and survival times before and after diagnosis of hepatic metastases. Results: H19 expression was found in the hepatic metastases of 64 of 80 patients. High expression (higher staining grades) of H19 in the metastases was found in 43 of 80 patients. However, H19 expression status in the hepatic metastases did not correlate with either the length of time to development of metastasis or overall survival. Conclusions: H19 is highly expressed in more than half of hepatic metastases derived from a range of carcinomas. Thus, these metastases may be suitable candidates for H19 DNA based treatment. Further studies are needed to determine whether H19 expression has prognostic value in metastatic liver disease using larger numbers of specific subtypes of primary carcinomas.

Unique expression patterns of H19 in human testicular cancers of different etiology

The expression pattern of the imprinted human H19 gene was investigated in testicular cancers of different etiology, as well as in normal testicular parenchyma, parenchyma without germ cells, and adjacent to testicular germ cell tumors of adolescents and adults (TGCTs), using RNase protection analysis, mRNA in situ hybridization and reverse-transcription polymerase chain reaction. While different total expression levels were detected in spermatocytic seminomas, lymphomas, a Sertoli cell tumor and Leydig cell tumors, none showed a disturbance of monoallelic expression. Strikingly, the majority of invasive TGCTs revealed expression of both parental alleles. The total level of expression highly correlated with differentiation lineage and stage of maturation, similar to that as reported during early normal embryogenesis. Biallelic expression could also be determined specifically in testis parenchyma containing the preinvasive lesion of this cancer. We therefore conclude that within the adult testis, biallelic H19 expression is specific for TGCTs, and that the level of expression is dependent on differentiation lineage and maturation stage. This is in agreement with the proposed primordial germ cell-origin of this cancer, and might be related to retention of embryonic characteristics in TGCTs. In addition, our data argue against H19 being a tumor suppressor gene.

The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene?

H19 is a paternally imprinted gene with unknown function. It is located in close proximity to the maternally imprinted IGF-2 gene on chromosome 11p15.5. In this study no consistent relationship between the expression of these two genes in clones derived from JEG-3 and JAr cell lines could be detected. Nor could a consistent relationship be detected between the expression levels of these two genes and between certain characteristic tumorigenic properties of these clones. We included in this study clones, expressing low H19 levels, which after transfection with an H19 expression construct highly expressed the H19 gene. In tumors, formed by the injection of cells of JAr or JEG-3 clones into nude mice, the H19 expression was high and irrelevant to the expression level in the cells before the injection. The same phenomenon was found for IGF-2 expression during tumorigenesis caused by cells of different JEG-3 clones and in some but not all JAr derived clones. Both H19 and IGF-2 are biallelicly expressed in all the JAr and JEG-3 clones. In summary, our observations point to the conclusion that H19 is not a tumor suppressor gene. However, its high expression in all the tumors formed after injection of cells of the JAr and JEG-3 clones, leaves its role, if any, in choriocarcinogenesis an open question.

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.