Kathryn E. Hacker

Variation in chromatin accessibility in human kidney cancer links H3K36 methyltransferase loss with widespread RNA processing defects.

Comprehensive sequencing of human cancers has identified recurrent mutations in genes encoding chromatin regulatory proteins. For clear cell renal cell carcinoma (ccRCC), three of the five commonly mutated genes encode the chromatin regulators PBRM1, SETD2, and BAP1. How these mutations alter the chromatin landscape and transcriptional program in ccRCC or other cancers is not understood. Here, we identified alterations in chromatin organization and transcript profiles associated with mutations in chromatin regulators in a large cohort of primary human kidney tumors. By associating variation in chromatin organization with mutations in SETD2, which encodes the enzyme responsible for H3K36 trimethylation, we found that changes in chromatin accessibility occurred primarily within actively transcribed genes. This increase in chromatin accessibility was linked with widespread alterations in RNA processing, including intron retention and aberrant splicing, affecting ∼25% of all expressed genes. Furthermore, decreased nucleosome occupancy proximal to misspliced exons was observed in tumors lacking H3K36me3. These results directly link mutations in SETD2 to chromatin accessibility changes and RNA processing defects in cancer. Detecting the functional consequences of specific mutations in chromatin regulatory proteins in primary human samples could ultimately inform the therapeutic application of an emerging class of chromatin-targeted compounds.

Meta-analysis of Clear Cell Renal Cell Carcinoma Gene Expression Defines a Variant Subgroup and Identifies Gender Influences on Tumor Biology

BACKGROUND Clear cell renal cell carcinoma (ccRCC) displays molecular and histologic heterogeneity. Previously described subsets of this disease, ccA and ccB, were defined based on multigene expression profiles, but it is unclear whether these subgroupings reflect the full spectrum of disease or how these molecular subtypes relate to histologic descriptions or gender. OBJECTIVE Determine whether additional subtypes of ccRCC exist and whether these subtypes are related to von Hippel-Lindau (VHL) inactivation, hypoxia-inducible factor (HIF) 1 and 2 expression, tumor histology, or gender. DESIGN, SETTING, AND PARTICIPANTS Six large, publicly available ccRCC gene expression databases were identified that cumulatively provided data for 480 tumors for meta-analysis via meta-array compilation. MEASUREMENTS Unsupervised consensus clustering was performed on the meta-arrays. Tumors were examined for the relationship of multigene-defined consensus subtypes and expression signatures of VHL mutation and HIF status, tumor histology, and gender. RESULTS AND LIMITATIONS Two dominant subsets of ccRCC were observed. However, a minor third cluster was revealed that correlated strongly with a wild type (WT) VHL expression profile and indications of variant histologies. When variant histologies were removed, ccA tumors naturally divided by gender. This technique is limited by the potential for persistent batch effect, tumor sampling bias, and restrictions of annotated information. CONCLUSIONS The ccA and ccB subsets of ccRCC are robust in meta-analysis among histologically conventional ccRCC tumors. A third group of tumors was identified that may represent a new variant of ccRCC. Within definitively clear cell tumors, gender may delineate tumors in such a way that it could have implications regarding current treatments and future drug development.

VHL Type 2B Mutations Retain VBC Complex Form and Function

Background von Hippel-Lindau disease is characterized by a spectrum of hypervascular tumors, including renal cell carcinoma, hemangioblastoma, and pheochromocytoma, which occur with VHL genotype-specific differences in penetrance. VHL loss causes a failure to regulate the hypoxia inducible factors (HIF-1α and HIF-2α), resulting in accumulation of both factors to high levels. Although HIF dysregulation is critical to VHL disease-associated renal tumorigenesis, increasing evidence points toward gradations of HIF dysregulation contributing to the degree of predisposition to renal cell carcinoma and other manifestations of the disease. Methodology/Principal Findings This investigation examined the ability of disease-specific VHL missense mutations to support the assembly of the VBC complex and to promote the ubiquitylation of HIF. Our interaction analysis supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF. We additionally demonstrated that Type 2B mutant pVHL forms a remnant VBC complex containing the active members ROC1 and Cullin-2 which retains the ability to ubiquitylate HIF-1α. Conclusions Our results suggest that subtypes of VHL mutations support an intermediate level of HIF regulation via a remnant VBC complex. These findings provide a mechanism for the graded HIF dysregulation and genetic predisposition for cancer development in VHL disease.

Tumor suppressive activity of prolyl isomerase Pin1 in renal cell carcinoma.

Pin1 specifically recognizes and catalyzes the cis‐trans isomerization of phosphorylated‐Ser/Thr‐Pro bonds, which modulate the stability, localization, and function of numerous Pin1 targets involved in tumor progression. However, the role of Pin1 in cancer remains enigmatic as the gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under‐expressed in kidney cancer, we have explored its role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under‐expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may have a mild tumor suppressive role in ccRCC.

Receptor tyrosine kinase-like orphan receptor 2 (Ror2) expression creates a poised state of Wnt signaling in renal cancer

Background: Expression of Ror2 leads to increased tumorigenicity in RCC. Results: Ror2 expression stabilizes an increased pool of soluble β-catenin, which enhances the response to Wnt stimulation. Conclusion: Ror2 promotes basal and canonical Wnt3a ligand-enhanced β-catenin signaling in RCC. Significance: This work demonstrates a novel Ror2-dependent tiered state of canonical Wnt signaling, promoting basal signals and priming RCC cells for a heightened response to the ligand. Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.

Structure/function analysis of recurrent mutations in SETD2 protein reveals a critical and conserved role for a SET domain residue in maintaining protein stability and histone H3 Lys-36 trimethylation

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

Emerging molecular classification in renal cell carcinoma: implications for drug development

In the past decade, progress has been made in the development of targeted therapies for advanced renal cell carcinoma (RCC). However, as multiple therapeutic choices become available to clinicians, we currently lack effective indicators that allow physicians to choose the best treatment option for specific patients. For approved targeted therapies, potential molecules that could indicate drug effectiveness in a specific tumor follow naturally from both the therapeutic mechanism and the previously elucidated tumor biology. However, in advanced RCC, the use of these molecules as biomarkers for treatment selection has shown equivocal results and requires further investigation. In addition to looking at specific molecular targets, subclassification of tumors based on their molecular characteristics may also allow stratification of patients based on therapeutic benefits, providing information for treatment selection. Furthermore, the continued development of such tumor classification schemes will hopefully uncover other molecular targets that warrant development as future RCC therapies. The use of molecular classification of patients’ tumors for treatment selection will provide the opportunity to increase the effectiveness of currently available therapies for advanced RCC and to judiciously pursue promising options for future RCC therapies.

Detection of Upper Tract Urothelial Malignancies by Computed Tomography Urography in Patients Referred for Hematuria at a Large Tertiary Referral Center

OBJECTIVE To evaluate the age-stratified prevalence of upper tract urothelial malignancies diagnosed on computed tomography urography in a large cohort of patients referred for initial evaluation of hematuria. MATERIALS AND METHODS A total of 1123 consecutive adults without a history of urothelial cancer underwent initial computed tomography urography for gross hematuria (n = 652), microscopic hematuria (n = 457), or unspecified hematuria (n = 14) at a single institution from October 2006 to October 2012. Imaging findings suggestive of urothelial lesions were correlated with clinical information, including cystoscopy, cytology, and surgical pathology reports. Patients subsequently diagnosed with urothelial cancer following a normal radiographic evaluation were identified and analyzed. Age, gender, smoking history, and location and type of malignancy were analyzed. RESULTS Upper tract urothelial cancer was detected in 4 (0.36%) patients, with a mean age of 66.5 years. All 4 patients presented with gross hematuria and were current or former smokers. None of the 535 patients under age 55 who underwent computed tomography urography were diagnosed with upper tract disease regardless of age, smoking history, or degree of hematuria. Likewise, no upper tract cancers were detected in patients referred for microscopic hematuria, regardless of age. CONCLUSION Detection of upper tract urothelial cancer by computed tomography urography is exceedingly rare in patients presenting at a tertiary referral center with hematuria, particularly in the lower risk strata (younger age, microscopic hematuria). Further investigation into risk-stratified approaches to imaging for hematuria workup is warranted to minimize unnecessary costs and radiation exposure.

Engaging Stakeholders and Patient Partners

Recent advances in engagement of stakeholders and patient partners in clinical research have bridged the disconnect between researchers and stakeholders, resulting in improved research goals with relevant outcomes, increased clinical trial enrollment, and improved communication of research results. This article focuses on the mechanisms, challenges, and benefits of patient and stakeholder engagement, with strategies for improvement. The 3 stages of clinical research and key iterative steps to create a reciprocal relationship are presented. Despite recent advances in stakeholder engagement, additional investigation and improved reporting of methods will facilitate strong reciprocal relationships between researchers and stakeholders.

Von Hippel-Lindau status influences phenotype of liver cancers arising from PTEN loss.

Background PTEN loss contributes to the development of liver diseases including hepatic steatosis and both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The factors that influence the penetrance of these conditions are unclear. We explored the influence of sustained hypoxia signaling through co-deletion of Pten and Vhl in a murine model. Methods We used a CreER-linked Keratin 18 mouse model to conditionally delete Pten, Vhl or both in somatic cells of adult mice, evaluating the resultant tumors by histology and gene expression microarray. Existing sets of gene expression data for human HCC and CC were examined for pathways related to those observed in the murine tumors, and a cohort of human CC samples was evaluated for relationships between HIF-1α expression and clinical outcomes. Results Both Pten deletion genotypes developed liver tumors, but with differing phenotypes. Pten deletion alone led to large hepatic tumors with widespread hepatosteatosis. Co-deletion of Pten and Vhl with the Keratin 18 promoter resulted in reduced steatosis and a reduced tumor burden that was characterized by a trabecular architecture similar to CC. Genes associated with hepatic steatosis were coordinately expressed in the human HCC dataset, while genes involved in hypoxia response were upregulated in tumors from the human CC dataset. HIF-1α expression and overall survival were examined in an independent cohort of human CC tumors with no statistical differences uncovered. Conclusion Pten deletion in Keratin 18 expressing cells leads to aggressive tumor formation and widespread steatosis in mouse livers. Co-deletion of Vhl and Pten results in lower tumor burden with gene expression profiling suggesting a switch from a profile of lipid deposition to an expression profile more consistent with upregulation of the hypoxia response pathway. A relationship between tumor hypoxia signaling and altered hepatic steatotic response suggests that competing influences may alter tumor phenotypes.