Posts Tagged ‘tumour’

Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Oncogene ID4

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Genome-scale studies of cancer samples have begun to provide a global depiction of genetic alterations in human cancers, but the complexity and volume of data that emerge from these efforts have made dissecting the underlying biology of cancer difficult, and little is known about the functions of most of the candidates that emerge. For example, in studies of 489 primary high-grade serous ovarian cancer genomes, 1825 genes were identified as targeted by recurrent amplification events. Systematic approaches to study the function of genes in cancer cell lines, such as genome-scale pooled short hairpin RNA (shRNA) screens, offer a means to assess the consequences of the genetic alterations found in such genome characterization efforts. The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance because many of these targets are not amenable to manipulation by small molecules or antibodies. RNA interference provides a direct way to study putative cancer targets; however, specific delivery of therapeutics to the tumor parenchyma remains an intractable problem.

Recently an shRNA-based approach was used to find genes that are both overexpressed in human primary tumors and essential for the proliferation of ovarian cancer cells. This approach identified 54 overexpressed and essential genes in ovarian cancer and 16 genes in non–small cell lung cancer that required further validation in vivo. Furthermore, many of these candidates represent targets that are not amenable to antibody-based therapeutics or traditional small molecule approaches. Thus, if one envisions a discovery pipeline that begins with cancer genomes and ends with novel therapeutics, there is a bottleneck at the point of in vivo validation of novel targets. Achieving silencing in the epithelial cells in the tumor parenchyma is especially critical to study the genetic alterations of interest. RNA interference (RNAi) is a potentially attractive means to silence the expression of candidate genes in vivo, particularly for undruggable gene products. However, systemic delivery of small interfering RNA (siRNA) to tumors has been challenging, owing to rapid clearance, susceptibility to serum nucleases, and endosomal entrapment of small RNAs, in addition to their inherent inadequate tumor penetration. Tumor penetration is also a problem for the delivery of siRNA and shRNA, among other cargos, and is characterized by limited transport into the extravascular tumor tissue beyond the perivascular region. This low penetration is thought to arise from the combination of dysfunctional blood vessels that are poorly perfused and a high interstitial pressure, especially in solid tumors, in part due to dysfunctional lymphatics. The leakiness of tumor vessels partially counteracts the poor penetration [the so-called enhanced permeability and retention (EPR) effect], but the size dependence and variability of this property can limit its usefulness. Desmoplastic stromal barriers can further impede transport of therapeutics through tumors. A new class of tumor-penetrating peptides has been described recently, which home to several types of tumors and leverage a consensus R/KXXR/K C-terminal peptide motif [the C-end rule (CendR)] to stimulate transvascular transport and rapidly deliver therapeutic cargo deep into the tumor parenchyma. These peptides are tumorspecific, unlike canonical cell-penetrating peptides (CPPs) that do not display cell- or tissue-type specificity, and are able to improve the delivery of small molecules, antibodies, and nanoparticles. Despite their promise, this class of peptides has not been successfully co-opted for siRNA delivery, in part owing to the additional challenges of delivering oligonucleotides across cell membranes, out of endosomes, and into the cytosol to achieve gene silencing. Here, an siRNA delivery vehicle has been designed that was tumorpenetrating and modular, so it could be easily assembled in a single step to accommodate different payloads to various genes of interest. It can be envisaged that such a technology would enable a platform wherein novel targets can be identified by structural and functional genomics and subsequently rapidly credentialed both in vitro and in vivo. Followup studies could then identify the mechanism of action underlying the observations and establish (and ultimately prioritize) novel oncogenes as therapeutic targets. To achieve this goal, a systematic effort was combined to identify genes that are both essential and genetically altered in human cancer cell lines and tumors with the development and deployment of a novel tumor-specific and tissue-penetrating siRNA delivery platform.

Current genome characterization efforts will eventually provide insight into the genetic alterations that occur in most cancers and may define new therapeutic targets. However, most epithelial cancers harbor hundreds of genetic alterations as a consequence of genomic instability. For example, whereas recurrent somatic alterations occur in a small number of genes in high-grade ovarian cancers, ovarian cancer genomes are characterized by multiple regions of copy number gain and loss involving at least 1825 genes. This genomic chaos complicates efforts to identify biologically relevant mutations critical for tumor maintenance.

To isolate which recurrent genetic alterations are involved in cancer initiation, tumor maintenance, and/or metastasis, functional assays can be performed after systematic manipulation of the candidate oncogenes. Results from Project Achilles was combined, a large scale screening effort to identify genes essential for proliferation and survival in human cancer cell lines with genome characterization of high-grade ovarian cancers. Using this approach, an oncogene candidate  was identified, ID4, which was amplified in 32% of high-grade serous ovarian cancers. ID4 is overexpressed in a large fraction of high-grade serous ovarian cancers, and ovarian cancer cell lines that overexpress ID4 are highly dependent on ID4 for survival and tumorigenicity. Expression of ID4 at levels corresponding to those observed in patient-derived samples induced transformation of immortalized ovarian and FT epithelial cells.

In summary, a targeted TPN was developed capable of precisely delivering siRNA into the tumor parenchyma, and have combined this technology with large-scale methods to credential ID4 as an oncogene target in ovarian cancer. Because large-scale efforts to characterize all cancer genomes accelerate, this capability illustrates a path to identify genes that are altered in tumors, validate those that are critical to cancer initiation and maintenance, and rapidly evaluate in vivo the subset of such genes amenable to RNAi therapies and clinical translation. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets.

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