The role of the oncofetal H19 lncRNA in tumor metastasis: orchestrating the EMT-MET decision.

Long non-coding RNA (lncRNA) genes are emerging as key players in the metastatic cascade. Current evidence indicate that H19 lncRNA and the microRNA(miRNA) miR-675, which is processed from it, play crucial roles in metastasis, through the regulation of critical events specifically the epithelial to mesenchymal (EMT) and the mesenchymal to epithelial transitions (MET). This review summarizes recent mechanistic pathways and tries to put together seemingly conflicting data from different reports under one proposed general scheme underlying the various roles of H19/miR-675 in the metastatic cascade. We propose several approaches to harnessing this knowledge for translational medicine.

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.

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.

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.

Evaluation of the 13C-octanoate breath test as a surrogate marker of liver damage in animal models.

BACKGROUND: Octanoate (also known as sodium octanoate), a medium-chain fatty acid metabolized in the liver, is a potential substrate for non-invasive breath testing of hepatic mitochondrial beta-oxidation. METHODS: We evaluated the 13C-octanoate breath test (OBT) for assessing injury in acute hepatitis and two rat models of liver cirrhosis, first testing octanoate absorption (per os or intraperitoneally (i.p.)) in normal rats. We then induced acute hepatitis with thioacetamide (300 mg/kg/i.p., 24-h intervals). Liver injury end points were serum aminotransferase levels and 13C-OBT (24 and 48 h following initial injection). Thioacetamide (200 mg/kg/i.p., twice per week, 12 weeks) was used to induce liver cirrhosis. OBT and liver histological assessment were performed every 4 weeks. Bile duct ligation (BDL) was used to induce cholestatic liver injury. We completed breath tests with 13C-OBT and 13C-methacetin (MBID), liver biochemistry, and liver histology in BDL and sham-operated rats (baseline, 6, 14, 20 days post-BDL). RESULTS: Octanoate absorbs well by either route. Peak amplitudes and cumulative percentage dose recovered at 30 and 60 min (CPDR30/60), but not peak time, correlated with acute hepatitis. Fibrosis stage 3 at week 8 significantly correlated with each OBT parameter. Cholestatic liver injury (serum bilirubin, ALP, gamma-GT, liver histology) was associated with significant suppression of the maximal peak values and CPDR30/60, respectively (P<0.05),using MBID but not 13C-octanoate. CONCLUSIONS: OBT is sensitive for potentially evaluating liver function in rat models of acute hepatitis and thioacetamide-induced liver cirrhosis but not in cholestatic liver injury. The MBID test may be better for evaluation of cholestatic liver disease in this model.

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.

Utility of a 13C-methacetin breath test in evaluating hepatic injury in rats.

BACKGROUND AND AIM: Methacetin is thought to be a good substrate for the evaluation of different cytochrome P450 enzymatic systems of liver microsomes because of its rapid metabolism and lack of toxicity in small doses. Recent studies indicate that a methacetin breath test may be a non-invasive alternative for the evaluation of liver function since it correlates well with the severity of liver damage. It may also discriminate between different stages of liver cirrhosis and correlates with the Child-Pugh score. The application of this test in experimental liver damage in animal models has not yet been examined. This study aimed to evaluate the efficacy of the (13)C-methacetin breath test in assessing the extent of hepatic injury in models of acute liver failure, liver cirrhosis, and fatty liver in rats. METHODS: Absorption of methacetin given per os or intraperitoneally in normal rats was evaluated. The association between liver mass and (13)C-methacetin breath test results was assessed in a 70% hepatectomy rat model. Fulminant hepatic failure was induced by three consecutive intraperitoneal injections of thioacetamide, 300 mg/kg, at 24 h intervals. For induction of liver cirrhosis, rats were given intraperitoneal injections of thioacetamide, 200 mg/kg, twice a week for 12 weeks. A methionine-choline deficient diet was used for the induction of fatty liver. Rats were analyzed for (13)C-methacetin by BreathID (MBID) using molecular correlation spectrometry. BreathID continuously sampled the animal's breath for 60 min and displayed the results on the BreathID screen in real-time. RESULTS: Methacetin was absorbed well irrespective of the administration method in normal rats. Liver mass was associated with peak amplitude, complete percent dose recovery (CPDR) at 30 and 60 min and MBID peak time. A high degree of association was also demonstrated with MBID results in acute hepatitis (peak amplitude, 19.6 +/- 3.4 vs 6.3 +/- 1.63.4; CPDR30, 6.0 +/- 3.3 vs 1.2 +/- 0.5; CPDR60, 13.3 +/- 4.5 vs 3.2 +/- 1.4; and peak time, 31.0 +/- 14.9 vs 46.9 +/- 10.8 min) and liver cirrhosis (peak amplitude, 24.4 +/- 2.3 vs 15.6 +/- 6.4; CPDR30, 7.9 +/- 1.2 vs 2.7 +/- 1.0; CPDR60, 17.8 +/- 2.6 vs 8.8 +/- 2.1; and peak time, 30.2 +/- 1.5 vs 59.6 +/- 14.5 min), but not with grade of liver steatosis. CONCLUSIONS: Methacetin is well absorbed and exclusively metabolized in the liver. MBID is a sensitive test and may be a useful tool for the evaluation of functional liver mass in animal models of acute liver failure and cirrhosis. However, MBID could not distinguish between fatty liver and normal liver in rats.

Fecal incontinence secondary to CAPD.

A 74-year-old patient who has developed fecal incontinence following continuous ambulatory peritoneal dialysis (CAPD) is presented. Incontinence was caused by elevated intra-abdominal pressure during peritoneal dialysis, which correlated with the volume of dialysate and position of the patient. The lowest pressure was found in the recumbent position. A change of dialysis schedule to continuous cycler peritoneal dialysis (CCPD) with "dry day" resulted in a disappearance of the symptoms.