Renal cell carcinoma deep sequencing: recent developments.

Renal cell carcinoma (RCC) is the most common type of renal cancer in adults. RCC is notoriously resistant to current therapies suggesting the need to improve our knowledge and create more effective therapies. The molecular genetic defects that occur in RCC are extensive and complex ranging from single DNA changes, to large chromosomal defects, to signature disruptions in the transcription of hundreds of genes. These changes are often shared within each histological RCC subtype, illustrating their significance to the disease phenotype. This review presents an overview of the genetic abnormalities that occur within the most common subtypes of RCC. We discuss the recent molecular findings that have advanced our understanding of the somatic architecture of renal tumors and their impact on disease therapeutics.

Species-specific difference in expression and splice-site choice in Inpp5b, an inositol polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe Syndrome.

The oculocerebrorenal syndrome of Lowe (OCRL; MIM #309000) is an X-linked human disorder characterized by congenital cataracts, mental retardation, and renal proximal tubular dysfunction caused by loss-of-function mutations in the OCRL gene that encodes Ocrl, a type II phosphatidylinositol bisphosphate (PtdIns4,5P(2)) 5-phosphatase. In contrast, mice with complete loss-of-function of the highly homologous ortholog Ocrl have no detectable renal, ophthalmological, or central nervous system abnormalities. We inferred that the disparate phenotype between Ocrl-deficient humans and mice was likely due to differences in how the two species compensate for loss of the Ocrl enzyme. We therefore turned our attention to Inpp5b, another type II PtdIns4,5P(2) 5-phosphatase encoded by Inpp5b in mice and INPP5B in humans, as potential compensating genes in the two species, because Inpp5b/INPP5B are the most highly conserved paralogs to Ocrl/OCRL in the respective genomes of both species and Inpp5b demonstrates functional overlap with Ocrl in mice in vivo. We used in silico sequence analysis, reverse-transcription PCR, quantitative PCR, and transient transfection assays of promoter function to define splice-site usage and the function of an internal promoter in mouse Inpp5b versus human INPP5B. We found mouse Inpp5b and human INPP5B differ in their transcription, splicing, and primary amino acid sequence. These observations form the foundation for analyzing the functional basis for the difference in how Inpp5b and INPP5B compensate for loss of Ocrl function and, by providing insight into the cellular roles of Ocrl and Inpp5b, aid in the development of a model system in which to study Lowe syndrome.

The tumor suppressor parafibromin is required for posttranscriptional processing of histone mRNA.

Parafibromin, encoded by the gene HRPT2, is a tumor suppressor protein associated with the RNA polymerase II-associated complex, Paf1 complex. HRPT2 mutations were first identified in patients with the multiple endocrine neoplasia syndrome, hyperparathyroidism-jaw tumor (HPT-JT) syndrome, and have also been found in sporadic parathyroid and renal tumors. However, the mechanisms by which parafibromin suppresses tumor formation remain unknown. In this study, we identify a novel role of parafibromin in the regulation of replication-dependent histones. Both in vitro and in vivo analyses reveal a posttranscriptional role of parafibromin in histone mRNA processing. Downregulation of parafibromin through RNA interference or in vivo mutations lead to uncleaved histone mRNA with polyadenylated tails. These results indicate that parafibromin regulates the 3' processing of histone RNA, an essential component of the cell cycle.

The E2F3-Oncomir-1 axis is activated in Wilms' tumor.

Oncomir-1 is an oncogenic cluster of microRNAs (miRNA) located on chromosome 13. Previous in vitro studies showed that it is transcriptionally regulated by the transcription factor E2F3. In this report, we combine expression profiling of both mRNA and miRNAs in Wilms' tumor (WT) samples to provide the first evidence that the E2F3-Oncomir-1 axis, previously identified in cell culture, is deregulated in primary human tumors. Analysis of RNA expression signatures showed that an E2F3 gene signature was activated in all WT samples analyzed, in contrast to other kidney tumors. This finding was validated by immunohistochemistry on the protein level. Expression of E2F3 was lowest in early-stage tumors and highest in metastatic tissue. Expression profiling of miRNAs in WT showed that expression of each measured member of the Oncomir-1 family was highest in WT relative to other kidney tumor subtypes. Quantitative PCR confirmed that these miRNAs were overexpressed in WT relative to normal kidney tissue. These results suggest that the E2F3-Oncomir-1 axis is activated in WT. Our study also shows the utility of integrated genomics combining gene signature analysis with miRNA expression profiling to identify protein-miRNA interactions that are perturbed in disease states.

Parafibromin, a component of the human PAF complex, regulates growth factors and is required for embryonic development and survival in adult mice.

Parafibromin, a transcription factor associated with the PAF complex, is encoded by the HRPT2 gene, mutations of which cause the hyperparathyroidism-jaw tumor syndrome (OMIM145001). To elucidate the function of parafibromin, we generated conventional and conditional Hrpt2 knockout mice and found that Hrpt2(-/-) mice were embryonic lethal by embryonic day 6.5 (E6.5). Controlled deletion of Hrpt2 after E8.5 resulted in apoptosis and growth retardation. Deletion of Hrpt2 in adult mice led to severe cachexia and death within 20 days. To explore the mechanism underlying the embryonic lethality and death of adult mice, mouse embryonic fibroblasts (MEFs) were cultured and Hrpt2 was deleted in vitro. Hrpt2(-/-) MEFs underwent apoptosis, while Hrpt2(+/+) and Hrpt2(+/-) MEFs grew normally. To study the mechanism of this apoptosis, Hrpt2(+/+) and Hrpt2(-/-) MEFs were used in cDNA microarray, semiquantitative reverse transcription-PCR, and chromatin immunoprecipitation assays to identify genes regulated by parafibromin. These revealed that Hrpt2 expression and the parafibromin/PAF complex directly regulate genes involved in cell growth and survival, including H19, Igf1, Igf2, Igfbp4, Hmga1, Hmga2, and Hmgcs2. Thus, our results show that expression of Hrpt2 and parafibromin is pivotal in mammalian development and survival in adults and that these functions are likely mediated by the transcriptional regulation of growth factors.

Parafibromin inhibits cancer cell growth and causes G1 phase arrest.

The HRPT2 (hereditary hyperparathyroidism type 2) tumor suppressor gene encodes a ubiquitously expressed 531 amino acid protein termed parafibromin. Inactivation of parafibromin predisposes one to the development of HPT-JT syndrome. To date, the role of parafibromin in tumorigenesis is largely unknown. Here, we report that parafibromin is a nuclear protein that possesses anti-proliferative properties. We show that overexpression of parafibromin inhibits colony formation and cellular proliferation, and induces cell cycle arrest in the G1 phase. Moreover, HPT-JT syndrome-derived mutations in HRPT2 behave in a dominant-negative manner by abolishing the ability of parafibromin to suppress cell proliferation. These findings suggest that parafibromin has a critical role in cell growth, and mutations in HRPT2 can directly inhibit this role.