Introduction

Reviewing brochures on the conventional cancer drugs in use today is undoubtedly a horrible and terrifying event. Seldom is any vital system in the body unaffected by a cancer drug, and sometimes they are seriously affected. Lack of tumor specificity is mostly responsible for these adverse events, which cause the therapeutic dose to be greatly limited. With few exceptions, these conventional therapeutic regimens also have narrow therapeutic windows; this is especially the case for locally advanced malignancies and for those that metastasize. Chemotherapy has resulted in a very modest survival gain and has not demonstrated a major impact on the downhill course of most common malignancies. Residual tumor cells that bypass even radical combinations of surgery, chemotherapy, and radiotherapy can lead to relapse, with an even more aggressive behavior. Moreover, the issue of the multidrug-resistant phenotype that some cancer cells acquire is a major obstacle . This frustrating state-of the-art behooves us to double our efforts in the search of innovative treatment avenues.

During the past decade, cancer gene therapy has been one of the most exiting areas of therapeutic research, and some research has led to advanced stages of clinical trials with promising outcomes. The clearest advantage for this targeted therapy approach is that normal tissue toxicity may be avoided if suitable targeted delivery and tumor-specific expression strategies are employed. The issue of very efficient targeted delivery, which is ultimately needed for successful cancer therapy, still poses a major challenge, and enormous effort has been made to overcome this challenge. Major advancements have been achieved with regard to the tumor-specific expression strategy, also called transcriptional targeting, as a result of great efforts to identify genes that are expressed specifically in tumors and marginally in normal cells. Different strategies to improve and design tumor-specific promoters have been reported . In this approach, the accumulated understanding of gene regulation has been exploited to target the diseased cells successfully with the therapeutic gene expression. Other approaches for targeting tumor cells specifically have also been developed. The transductional targeting approach involves the genetic modification of viral vectors to enhance its tropism to achieve selective delivery of transgenes to target cells, and it may also result in cancer cell lyses when using oncolytic viruses. To achieve maximal specificity, transcriptional targeting may be combined with transductional targeting. Targeted therapy also involves small-molecule drugs and monoclonal antibodies. In the latter case, toxic molecules can be delivered specifically to tumor cells through monoclonal antibodies by exploiting our knowledge of the unique antigens that are expressed on tumor cells . Moreover, cancer gene therapy approaches can be administered in combination with chemotherapy and radiotherapy.

This chapter focuses on the successful use of H19 gene regulatory sequences for transcriptional targeting of cancer cells. The heterologous H19 promoter is transactivated by the same transcription factors and complexes that drive its transcriptional upregulation in the course of malignancy. The next step after determining high expression levels of H19 in numerous types of cancer was to characterize H19’s regulatory sequences. In our group, cloning of different fragments of the human H19 promoter was done and enabled the examination of their transcriptional activity in a variety of cancer cell lines. The region located between −229 and −134 bp contains a sequence element(s) with a positive transcriptional activity. The region between 85 and −61 bp is rich in CpGs, and it plays a significant role in the regulation of H19 transcription. This region contains transcription factors binding sites, including the CCATT box, which binds transcription factors from the C/EBP family. The sequence region between −85 and −134 bp acts as a binding site for negative acting transcription factor(s) in carcinoma cells .

Therefore, expression of the therapeutic toxin—in our case, diphtheria toxin A driven by H19 promoter—is predicted to occur in a manner specific for tumor expressing or overexpressing H19 gene product. This approach has been applied in clinical trials in patients of bladder, ovarian, colon, liver metastases and pancreatic carcinomas with promising outcomes . Preclinical validation of this approach is under development for other types of human carcinomas.

Therapies that target the H19 RNA or that exploit its transcriptional regulatory element to drive the expression of a cytotoxic gene specifically to tumor cells would therefore be very valuable in treating tumors that have a high level of H19 RNA. However, the potential of this transcriptional targeting approach might be even greater if two different regulatory sequences, selected from the cancer-specific promoters H19, IGF2-P3, and IGF2-P4, are utilized in the same therapeutic construct because many tumors that lack H19 gene expression have been found to express IGF2. In a second approach, a single promoter may be used to drive two cytotoxic genes having synergistic effect on a single construct. Both approaches have shown superior tumor growth inhibition activity in preclinical studies of bladder cancer .

H19 and Tumorigenicity

Understanding the function of the H19 gene is crucial if we hope to uncover its roles in cancer and harness this knowledge for therapeutic benefit. The H19 gene was the first imprinted noncoding RNA gene to be discovered . It is highly expressed in most fetal organs but repressed immediately after birth . Regarding its role in tumorigenicity, accumulating data from our group and others support the oncogenic function of H19 RNA, although several groups have reported H19 as having a tumor suppressor activity . The H19 locus harbors the potential to produce various products that may account for this discrepancy. From H19 exon 1, miR675 is produced . However, this microRNA is predicted to have a tumor-promoting role by targeting retinoblastoma tumor suppressor . Moreover, a short-lived, approximately 120-kb-long antisense transcript called 91H is produced at the imprinted H19/IGF2 locus, overlapping the H19 gene. 91H transcript is stabilized in breast cancer cells and overexpressed in human breast tumors . Recently, an antisense transcript encoded by H19 has been identified and called HOTS (H19 opposite tumor suppressor); it is a protein coding transcript and has a probable tumor suppressor function .

H19 RNA is emerging as one of the key players in cancer biology. Although H19 was long ago reported to show aberrant expression in various types of human cancer, where the majority of human tumors frequently express the H19 gene, clear, causal evidence of its role in cancer has only recently come to light.

H19 RNA contributes significantly to several aspects of the malignant phenotype, including proliferation, hypoxic stress response, angiogenesis, metastasis, and multidrug resistance . Cancer cells devoid of H19 expression encounter a very significant retardation of tumor growth in vivo .

To shed light on H19’s mechanism of action, which remained obscure until recently, we and others utilized a strategy of identifying upstream effectors and also downstream targets by applying the global gene expression profiling to identify genes modulated by both H19 overexpression and knockdown. Consequently, our group and others identified numerous modulators of H19 gene expression, including chemical carcinogens (e.g., BBN and DEN); retinoic acid; steroid and peptide hormones; growth factors such as hepatocyte growth factor; transcription factors such as E2F, HIF1α, p53, and c-myc; and environmental factors such as hypoxia and serum starvation. Because a complete report is beyond the scope of this concise review, for in-depth discussion, readers are referred to references . Here, we discuss principal findings.

The identification of molecular mechanisms used to mediate the hypoxic cancer cellular response is of great interest to identify targets that might compromise the survival of hypoxic cells. Hypoxia is a major trigger for tumor angiogenesis, invasiveness, metastasis, chemoresistance, radioresistance, and loss of genomic stability. It is also associated with poor prognosis in some types of human cancers. Notably, most of these conditions are associated with an increase in the level of H19 RNA.

Recently, we uncovered a molecular mechanism that integrates H19, p53, and HIF1-α to hypoxic stress response . We demonstrated a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. p53 suppresses H19 elevation in hypoxic cancer cells. Furthermore, we identified HIF1-α as the factor responsible for H19 elevation under hypoxic stress in the tumor cells in which p53 is mutated .

Consequently, H19 functions as a stress modulator and a survival factor, and it is involved in several fundamental processes, including epithelial–mesenchymal transition (EMT), malignant transformation, cell cycle transition, metastasis, and neo-angiogenesis . EMT is an important process on the way to the malignant phenotype; notably, H19 upregulation occurs in the stroma as well as in the epithelium . In the metastatic tumor stage, which bears a striking similarity to the embryonic stage, H19 involvement appears to be essential: Adherent and cohesive cells lose their anchorage, migrate under stressful conditions to remote sites, and replicate with neovascular support. Thus, H19 seems to occupy a central role in the cancer embryonic shift .

Because hypoxia readily occurs in the majority of solid tumors driving critical steps in tumor development and metastasis and resistance to therapeutic modalities, placing the H19 gene product in this deadly circuit undoubtedly will have major impacts in its utility as a high-priority target for cancer gene therapy.

Targeting Toxins and Suicide Genes in Gene Therapy

By definition, a suicide gene has a product that will cause a cell to kill itself through apoptosis. This product can be transcribed by a variety of promoters (e.g., constitutive promoters and tissue-specific promoters), but tumor selectivity can be approached through the utility of tumor-specific promoters. The specificity of promoter-targeted therapy can also derive from cancer-specific conditions such as hypoxia . Inducible promoters affected, for example, by radiation, heat, and drugs have also been reported .

Finding a promoter that uniquely directs expression in cancer cells such as in the case of H19 is of great importance. Another example is the human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase—a critical factor for cell immortalization and tumorigenesis. As demonstrated by our group and others, the use of the hTERT promoter has provided targeted preclinical therapeutic results in bladder and hepatocellular carcinoma cells . Moreover, our group used promoters of IGF2 gene for this purpose .

The main safety advantage of specific promoters therapy is sometimes accompanied by the disadvantage of weak activity, consequently resulting in a decrease in therapeutic efficacy. To improve specific but weak promoters, enhancers can be added and negative regulatory elements can be removed. For example, by insertion of four tandem copies of the synthetic androgen responsive element, a nearly 20-fold enhancement of activity over the native PSA promoter and enhancer (PSE) was achieved . In the case of H19, this promoter has the great advantage of its specificity to cancerous cells and at the same time demonstrates strong promoter activity similar to SV40.

Targeted therapy also involves small-molecule drugs and monoclonal antibodies. Monoclonal antibodies that recognize, for example, the CD20 molecule (tositumomab and ¹³¹ I-tositumomab (Bexxar), and ibritumomab tiuxetan (Zevalin)) and CD30 molecules (brentuximab vedotin (Adcetris)) linked to toxic molecules have been approved by the U.S. Food and Drug Administration (FDA).

Experimental Studies OF DTA-H19

The investigational agent BC-819, also referred to throughout this chapter as DTA-H19, is a double-stranded DNA plasmid of 4560 bp in length that carries the gene for the DT-A chain under the regulation of the H19 promoter sequence. This is a targeted cancer therapy because DT-A chain expression is triggered by the presence of transcription factors that upregulate H19 RNA expression in tumor cells. The selective initiation of toxin expression results in selective tumor cell destruction via inhibition of protein synthesis in the tumor cell, enabling highly targeted cancer treatment. DTA-H19 is being developed for the treatment of cancers that have upregulated levels of H19 expression. The first four indications under development are transitional cell carcinoma (TCC) of the bladder, pancreatic cancer, ovarian cancer, and hepatic cancer (either primary hepatic cell carcinoma or hepatic metastases of other primary cancers). DTA-H19 is administered intravesically into the bladder of TCC patients after mixing it just prior to intravesical instillation with the cationic transfection agent polyethyleneimine (PEI) ( in vivo jetPEI) to enhance in vivo transfection efficiency. For other indications cited previously, DTA-H19 is given by local/regional routes of delivery (intratumorally, intraperitoneally, or by hepatic artery infusion (HAI)), and in these cases it is not previously mixed with PEI but injected as naked DNA. In all clinical trials, patients’ tumors must be tested and be positive for H19 RNA in order to be eligible for treatment.

Nonclinical Pharmacology

Nonclinical studies ( in vitro/in vivo ) have been performed to demonstrate the potential efficacy of BC-819 in the different targeted cancers (bladder cancer, pancreatic cancer, ovarian cancer, and hepatic metastases) having upregulated levels of H19 expression.

In vitro pharmacology studies started by analyzing the expression of the H19 gene in patients’ tumor tissues and in human tumor cell lines. Then, the selective expression of a reporter gene (luciferase) and/or the DT-A chain controlled by the H19 gene promoter was assessed in vitro at different human cancer cell lines expressing H19. Finally, the efficacy of BC-819 in terms of protein synthesis inhibition was assessed in tumor cell lines of various origins.

Once a proof of concept of efficacy was obtained in vitro , in vivo nonclinical studies were conducted in several animal models with BC-819 administered as a naked DNA (for ovarian, hepatic metastases and pancreatic cancer) and in a complex suspension with PEI (for bladder cancer).

In Vitro Nonclinical Pharmacology

Bladder Cell Lines

The cell-specific activity of the DT-A chain driven by the H19 promoter region was demonstrated in human bladder carcinoma cells (T24P) and human fibroblast cells (IMR-90). There was dose-dependent inhibition of luciferase expression (the reporter gene) in T24P cells starting at 0.1 μg of co-transfected BC-819 and complete inhibition with 1.0 μg. This demonstrated that luciferase production was inhibited in cells by the expression of the DT-A chain under the regulation of the H19 promoter only in cells that express H19 RNA (i.e., T24P bladder cancer cells) and not in cells that do not express H19 RNA (i.e., the IMR-90 fibroblast cell line). It was further demonstrated that the inhibition of luciferase expression is indeed attributable to the toxic effects of the DT-A chain because this effect was not observed in 293-DT-A-resistant cells .

Pancreatic Carcinoma Cells

Two human pancreatic carcinoma cell lines expressing high levels of H19 (CRL 2547 and CRL 2119) were co transfected with 2 μg of LucSV40 and different concentrations of BC-819 vector. This experiment demonstrated that DT-A expression resulted in successful inhibition of protein synthesis as reflected by the decrease of luciferase activity in pancreatic tumor cells cell lines expressing H19 .

H19 Expression in Pancreatic Tumors

Pancreatic tumors from 36 patients and normal distal pancreatic tissue of 7 patients with tumors (obtained from the pathology department at Hadassah Medical Center, Israel) were examined for H19 expression by in situ hybridization (ISH) and/or polymerase chain reaction (PCR). Of the 36 tumors tested, 70% were positive for H19 expression.

According to the results obtained from the biopsies of the 16 patients with unresectable pancreatic cancer screened in the phase I/IIa study, 90% of the patients showed H19 expression in the tumor. A representative example of ISH of a pancreatic tumor is shown in Figure 8.1 . No H19 expression was found in normal pancreatic tissue distal to tumor regions .

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