Abraham Czerniak

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.

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.

Role of endoscopic retrograde cholangiopancreatography in differentiating pancreatic cancer coexisting with chronic pancreatitis

The pancreatographic appearance and the clinical presentation of ten patients presenting with adenocarcinoma of the head of the pancreas coexisting with chronic pancreatitis were compared with those of 45 patients with chronic pancreatitis (CP), without malignancy, investigated at the same time period. All ten patients, had typical pancreatographic findings of CP, combined with an elongated narrowing of the duct of Wirsung. Marked localized irregularity of the adjacent main duct and of side branches were found in all ten patients. Such findings were not detected in the other 45 patients with CP only. Ultrasonography or computed tomography have detected a definite pancreatic mass in only five of these patients, and in six patients with CP without malignancy. It is concluded that endoscopic retrograde cholangiopancreatography is highly accurate in detecting pancreatic cancer coexisting with CP. It is primarily helpful in elderly patients having severe degrees of CP to rule out cancer.

Early Endoscopic Sphincterotomy in the Management of Acute Gallstone Pancreatitis in Elderly Patients

Eighteen elderly patients with acute attacks of gallstone pancreatitis underwent early endoscopic sphincterotomy of the papilla of Water. Eleven patients were considered to be at high risk for surgery due to chronic cardiorespiratory or renal problems. The outcome of these patients was compared with that of 20 consecutive elderly patients with gallstone pancreatitis treated at the same time by means other than endoscopic sphincterotomy. Endoscopic sphincterotomy resulted in an immediate clinical improvement in all patients, except in one patient who developed transient cholangitis; there was no mortality. In contrast, there was one death (5%) and 20% morbidity in the controls. Mean hospitalization period was shorter in patients undergoing sphincterotomy (6 compared with 9.5 days), although the patients managed by sphincterotomy were initially more seriously ill than controls. Only two of the 11 high‐risk patients underwent elective cholecystectomy; all others were well during a mean follow‐up of 22 months. It is concluded that early endoscopic sphincterotomy is highly effective and safe in acute attacks of gallstone pancreatitis in elderly high‐risk patients.

The feasibility of in vivo resection of the left lobe of the liver and its use for transplantation

The anatomical possibility of resecting the left lobe of the liver (segments II and III) in living subjects and using it for transplantation was evaluated. A group of 60 cadaveric livers were dissected at autopsy. The vascular and biliary elements of the left lobe were isolated and the lobe was resected and evaluated for possible grafting. The left lobe was 12–28% (mean 19.4%) of the liver mass. An extrahepatic segment of the left hepatic vein was isolated in 95% of specimens. Arterial blood supply to the left lobe consisted of a single artery (92%) or two arteries (8%). A single portal vein segment to the left lobe (type I) was: found in 35% livers. Portal vein branches originated from a common orifice (type II, 35%) or separately (type III, 30%) from the left portal vein, and in these instances, preparation of a portal segment necessitated partial section of the left portal vein wall. Biliary drainage was extrahepatic in 56 livers and consisted of a single duct (type I, 78%), or two ducts (type II, 15%). The resected left lobe was evaluated as satisfactory (single hepatic vein and artery, types I or II portal vein, type I bile duct) in 48% of cases, while a less-satisfactory lobe (type III portal vein or type II bile duct) was obtained in 33%. It was found anatomically difficult or impossible to resect the left lobe for possible transplantation in 11 (19%) liver specimens.

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.

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.

Combined Systemic Chronotherapy and Hepatic Artery Infusion for the Treatment of Metastatic Colorectal Cancer Confined to the Liver

Background: The optimal treatment of patients with metastatic colorectal cancer is still a clinical challenge. We describe the use of combined hepatic arterial infusion (HAI) of irinotecan (CPT-11) in conjunction with systemic chronotherapy infusion of 5-fluorouracil (5FU), folinic acid and carboplatin in patients with colorectal liver metastases. Methods: Twenty-three patients with colorectal cancer and isolated liver metastases were enrolled in this trial. Intraoperative insertion of an intra-arterial catheter into the hepatic artery was accomplished during the colon operation (in cases of synchronous tumor) or as a separate procedure in colorectal cancer patients with newly diagnosed liver metastases. A systemic double-lumen double-chamber port was inserted via the subclavian vein as a separate procedure. The treatment plan included irinotecan given by intra-arterial infusion at 150 mg/m2 for 1 h. After 2 weeks of rest chronomodulated 5FU (700 mg/m2; peak delivery rate at 04:00 h), leucovorin (175 mg/m2; peak delivery rate at 04:00 h) and carboplatin (40 mg/m2; peak delivery rate at 16:00 h) for 4 days was followed by 10 days’ rest and then given again. After 10 days’ rest another HAI was introduced using the same method. Each cycle of therapy included 2 HAI courses and 2 chronotherapy courses in between. After 2 complete cycles, patients were evaluated for their response with weekly accessed toxicity recording. Results: Seven women, 8 men, median age 61 years (range 46–72). Eight patients had synchronous colon and hepatic disease and 7 patients had metachronous disease. Ten patients had previously been treated with 5FU and leucovorin while 5 patients were chemonaive. The mean number of cycles were 11.6 per patient (range 8–19). Partial response was achieved in 6 patients (40%) and was followed by laparoscopic radiofrequency ablation in 5 patients (33%). Disease stabilization was observed in 2 patients (13%) and disease progression in 7 patients (47%) mainly after previous chemotherapy failure. Side effects were infrequent and mild including grade 2 GIT complaints (5 patients), RUQ pain during HAI (9 patients) and grade 2 hematological complaints in 2 patients. Conclusion: A combined chemotherapy protocol (HAI and chronotherapy) with irinotecan (CPT-11) together with chronomodulated infusion of 5FU, folinic acid and carboplatin can be used in metastatic colorectal patients with a high efficacy rate and minor side effects especially in pretreated patients.