A phase 1/2a, dose-escalation, safety, pharmacokinetic, and preliminary efficacy study of intraperitoneal administration of BC-819 (H19-DTA) in subjects with recurrent ovarian/peritoneal cancer.

BACKGROUND: H19 is a paternally imprinted, oncofetal gene expressed in various embryonic tissues and in 85% of the ovarian tumors. H19-DTA (BC-819) is a DNA plasmid that drives the expression of the diphtheria toxin gene under the regulation of the H19 promoter sequence and therefore is a potential treatment for various tumors that overexpress the H19 gene, among them-ovarian cancer. OBJECTIVE: To assess the safety and efficacy of intra-peritoneal (IP) instillations of H19-DTA (BC-819) plasmid in treating ovarian/peritoneal cancer patients with advanced recurrent disease. METHODS: A phase 1-2A multi-centric trial included 14 eligible patients who were either platinum-refractory or platinum-resistant with positive H19 expression. Patients were treated IP with escalating weekly doses of BC-819 for a maximum of 6-9 weeks. Dose-limiting toxicities (DLT) were assessed after the first course of treatment for each patient and each subsequent cohort was enrolled once each subject had completed the first course of treatment and its 4-week follow-up period. The occurrence of adverse events (AEs) and response to treatment were assessed after the induction course and then periodically. RESULTS: During the study, no DLTs were observed. Only 5 grade 1 and 2 AEs, which occurred in 4 patients were considered as possibly related to BC-819. The best tumor response seen was stable disease. Median survivals of 3.2, 5.3 and 6.5 months were observed for the 60, 120 and 240 mg cohorts, respectively. CONCLUSIONS: BC-819 can be considered safe and well tolerated in intraperitoneal doses up to 240 mg. Hybridization of intraperitoneal chemotherapy with the biological treatment of BC-819 should be further evaluated in phase 2 and 3 studies.

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.

The H19 Long non-coding RNA in cancer initiation, progression and metastasis - a proposed unifying theory.

The imprinted oncofetal long non-coding RNA (lncRNA) H19 is expressed in the embryo, down-regulated at birth and then reappears in tumors. Its role in tumor initiation and progression has long been a subject of controversy, although accumulating data suggest that H19 is one of the major genes in cancer. It is actively involved in all stages of tumorigenesis and is expressed in almost every human cancer. In this review we delineate the various functions of H19 during the different stages in the complex process of tumor progression. H19 up-regulation allows cells to enter a "selfish" survival mode in response to stress conditions, such as destabilization of the genome and hypoxia, by accelerating their proliferation rate and increasing overall cellular resistance to stress. This response is tightly correlated with nullification, dysfunction or significant down-regulation of the master tumor suppressor gene P53. The growing evidence of H19's involvement in both proliferation and differentiation processes, together with its involvement in epithelial to mesenchymal transition (EMT) and also mesenchymal to epithelial transition (MET), has led us to conclude that some of the recent disputes and discrepancies arising from current research findings can be resolved from a viewpoint supporting the oncogenic properties of H19. According to a holistic approach, the versatile, seemingly contradictory functions of H19 are essential to, and differentially harnessed by, the tumor cell depending on its context within the process of tumor progression.

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.

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.

A chromatin immunoprecipitation screen in mouse keratinocytes reveals Runx1 as a direct transcriptional target of DeltaNp63.

Development of the skin epidermis and appendages such as hair follicles involves coordinated processes of keratinocyte proliferation and differentiation. The transcription factor p63 plays a critical role in these steps as evident by a complete lack of these structures in p63 null mice. The p63 gene encodes for two proteins TAp63 and DeltaNp63, the latter being the more prevalent and dominant isoform expressed in keratinocytes. Although numerous p63 target genes have been identified, these studies have been limited to transformed human keratinocyte cell lines. Here, we have employed a genomic screening approach of chromatin immunoprecipitation (ChIP) coupled with an enrichment strategy to identify DeltaNp63 response elements in primary mouse keratinocytes. Analysis of p63-ChIP-derived DNA segments has revealed more than 100 potential target genes including novel as well as mouse counterparts of established human p63 targets. Among these is Runx1, a transcription factor important for hair follicle development. We demonstrate that DeltaNp63 binds to a p63-response element located within a well-conserved enhancer of the Runx1 gene. Furthermore, siRNA mediated reduction of DeltaNp63 in mouse keratinocytes reduces Runx1 expression. Consistent with this, endogenous Runx1 levels are lower in the skin of p63(+/-) animals as compared to wild type animals. Lastly, we demonstrate that DeltaNp63 and Runx1 are co-expressed in specific compartments of the hair follicle in a dynamic fashion. Taken together our data demonstrate that p63 directly regulates Runx1 gene expression through a novel enhancer element and suggests a role for these two transcription factors in dictating skin keratinocyte and appendage development.

Dynamic expression of Runx1 in skin affects hair structure.

The three mammalian Runx transcription factors, some of which are known to be involved in human genetic diseases and cancer, are pivotal players in embryo development and function as key regulators of cell fate determination and organogenesis. Here, we report the expression of Runx1 during the development of hair and other skin appendages in the mouse and describe the effect of Runx1 on the structural hair output. In hair follicles, where the three Runx proteins are expressed, Runx1 expression is most prominent in both mesenchymal and epithelial compartments. The epithelial expression includes the hair keratin forming layers of the hair shaft and the bulge, where interestingly, Runx1 is co-expressed with keratin 15, a putative hair follicle stem cell marker. In the hair mesenchyme, during early stages of hair morphogenesis, Runx1 is expressed in a discrete dermal sub-epithelial layer, while at later stages it is found in a hair cycle dependent pattern in the dermal papilla. To elucidate the function of Runx1 in the hair follicle we have generated a Runx1 epidermal conditional knockout and found that the mutant mice display a remarkable structural deformation of the zigzag hair type. The data delineate Runx1 as a novel specific marker of several hair follicle cell types and sheds light on its role in hair morphogenesis and differentiation.

Runx3 is involved in hair shape determination.

Transcriptional regulators of the Runx family play critical roles in normal organ development and, when mutated, lead to genetic diseases and cancer. Runx3 functions during cell lineage decisions in thymopoiesis and neurogenesis and mediates transforming growth factor-beta signaling in dendritic cells. Here, we study the function of Runx3 in the skin and its appendages, primarily the hair follicle, during mouse development. Runx3 is expressed predominantly in the dermal compartment of the hair follicles as they form and during the hair cycle, as well as in the nail and sweat gland skin appendages. Distinct expression is also detected periodically in isolated cells of the epidermis and in melanocytes, populating the hair bulb. Runx3-deficient mice display a perturbation of the normal hair coat, which we show to be due to hair type and hair shape changes. Thus, one of the functions of Runx3 in skin may be to regulate the formation of the epithelial derived structural hair by affecting dermal to epidermal interactions.