Tatiana Birman

The imprinted H19 gene is a marker of early recurrence in human bladder carcinoma

Aims—To investigate the expression of the imprinted oncofetal H19 gene in human bladder carcinoma and to examine the possibility of using it as a tumour marker, similar to other oncofetal gene products. Methods—In situ hybridisation for H19 RNA was performed on 61 first biopsies of bladder carcinoma from Hadassah Medical Centre in Jerusalem. The intensity of the reaction and the number of tumour cells expressing H19 in each biopsy were evaluated in 56 patients, excluding biopsies with carcinoma in situ. The medical files were searched for demographic data and disease free survival. Results—More than 5% of cells expressed H19 in 47 of the 56 (84%) biopsies. There was a decrease in the number of cells expressing H19 with increasing tumour grade (loss of differentiation) (p = 0.03). Disease free survival from the first biopsy to first recurrence was significantly shorter in patients with tumours having a larger fraction of H19 expressing cells, controlling for tumour grade. This was also supported by the selective analysis of tumour recurrence in patients with grade I tumours. Conclusions—It might be possible to use H19 as a prognostic tumour marker for the early recurrence of bladder cancer. In addition, for the gene therapy of bladder carcinoma that is based on the transcriptional regulatory sequences of H19, the expression of H19 in an individual biopsy could be considered a predictive tumour marker for selecting those patients who would benefit from this form of treatment.

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.

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.

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.

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.

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.

Inhibition of tumor growth by DT-A expressed under the control of IGF2 P3 and P4 promoter sequences.

The human IGF2 P3 and P4 promoters are highly active in a variety of human cancers. We here present an approach for patient oriented therapy of TCC bladder carcinoma by driving the diphtheria toxin A-chain (DT-A) expression under the control of the IGF2 P3 and P4 promoter regulatory sequences. High levels of IGF2 mRNA expression from P3, P4 or both promoters were detected in 18 TCC samples (n = 29) by ISH or RT-PCR. Normal bladder samples (n = 4) showed no expression from either promoter. The activity and specificity of the IGF2 P3 and P4 regulatory sequences were established in human carcinoma cell lines by means of luciferase reporter gene assay. These sequences were used to design DT-A expressing, therapeutic vectors (P3-DT-A and P4-DT-A). The activity of both was determined in cell lines (in vitro) and the activity of P3-DT-A was determined in a heterotopic animal model (in vivo). The treated cell lines highly responded to the treatment in a dose-response manner, and the growth rate of the developed tumors in vivo was highly inhibited (70%) after intratumoraly injection with P3-DT-A compared to non-treated tumors (P < 0.0002) or tumors treated by luciferase gene expressing LucP3 vector (P < 0.002).

Adult Hypertension in Intrauterine Growth-Restricted Offspring of Hyperinsulinemic Rats Evidence of Subtle Renal Damage

In humans, intrauterine growth-restricted newborns are prone to develop hypertension as adults. We studied a rat model of pregnancy-induced hypertension associated with intrauterine growth restriction (IUGR) produced by chronic administration of insulin. Fetuses of hyperinsulinemic dams (HDs) were smaller than those of normal dams (5.1±0.4 g versus 5.6±0.1 g, respectively; P<0.05). At 16 weeks of age, tail-cuff systolic blood pressure was measured, the rats were placed in metabolic cages and euthanized, and the kidneys were examined. Male but not female offspring of HDs (n=9) had higher blood pressure than normal-pregnancy offspring (n=12; 148±11 mm Hg versus 118±14 mm Hg; P<0.004). In contrast to other models, there was no difference in ours in the number and volume of glomeruli. However, there were significantly greater glomerular, tubulointerstitial, and vascular damage indices in the kidneys of male HD offspring versus controls (2.01±0.34 versus 1.08±0.16, 1.80±0.34 versus 0.76±0.12, and 2.13±0.81 versus 0.78±0.16, respectively; P<0.0001), with similar tubulointerstitial findings in females. Increased expression of collagen type IV, a kidney damage marker indicating fibrosis, was found in the tubulointerstitium. This may be associated with downregulation of bone morphogenetic protein 6, a presumptive antifibrogenic agent, at the end of gestation. In conclusion, male offspring of HDs displayed IUGR and adult hypertension accompanied by several indices of renal fibrosing damage, mainly in the renal tubulointerstitium. Our findings suggest that there is >1 pathway of fetal programming leading from IUGR to development of hypertension in later life.

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.

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.