Jodi K. Maranchie

ClearCode34: A Prognostic Risk Predictor for Localized Clear Cell Renal Cell Carcinoma

BACKGROUND Gene expression signatures have proven to be useful tools in many cancers to identify distinct subtypes of disease based on molecular features that drive pathogenesis, and to aid in predicting clinical outcomes. However, there are no current signatures for kidney cancer that are applicable in a clinical setting. OBJECTIVE To generate a signature biomarker for the clear cell renal cell carcinoma (ccRCC) good risk (ccA) and poor risk (ccB) subtype classification that could be readily applied to clinical samples to develop an integrated model for biologically defined risk stratification. DESIGN, SETTING, AND PARTICIPANTS A set of 72 ccRCC sample standards was used to develop a 34-gene classifier (ClearCode34) for assigning ccRCC tumors to subtypes. The classifier was applied to RNA-sequencing data from 380 nonmetastatic ccRCC samples from the Cancer Genome Atlas (TCGA), and to 157 formalin-fixed clinical samples collected at the University of North Carolina. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Kaplan-Meier analyses were performed on the individual cohorts to calculate recurrence-free survival (RFS), cancer-specific survival (CSS), and overall survival (OS). Training and test sets were randomly selected from the combined cohorts to assemble a risk prediction model for disease recurrence. RESULTS AND LIMITATIONS The subtypes were significantly associated with RFS (p<0.01), CSS (p<0.01), and OS (p<0.01). Hazard ratios for subtype classification were similar to those of stage and grade in association with recurrence risk, and remained significant in multivariate analyses. An integrated molecular/clinical model for RFS to assign patients to risk groups was able to accurately predict CSS above established, clinical risk-prediction algorithms. CONCLUSIONS The ClearCode34-based model provides prognostic stratification that improves upon established algorithms to assess risk for recurrence and death for nonmetastatic ccRCC patients. PATIENT SUMMARY We developed a 34-gene subtype predictor to classify clear cell renal cell carcinoma tumors according to ccA or ccB subtypes and built a subtype-inclusive model to analyze patient survival outcomes.

Clinical implications of hypoxia inducible factor in renal cell carcinoma

Management of renal cell carcinoma (RCC) has made considerable strides in the past decade, due in large part to identification of the von Hippel Lindau (VHL) tumor suppressor as a negative regulator of hypoxia inducible factor alpha (HIF-alpha) protein expression. Stabilization of HIF-alpha appears to be critical for renal tumorigenesis, and is observed even in VHL-independent RCC. Thus, an understanding of the pathways that regulate expression and activation of the different HIF-alpha isoforms is key to delineating the mechanism of renal transformation and for the development of novel therapeutics. A number of agents targeting HIF-alpha or its transcriptionally-regulated genes have shown promise in treatment of RCC. However, more effective treatment strategies are still needed. This report provides a directed review of recent discoveries defining the role of HIF in renal tumorigenesis and their relevance to the clinical advances in targeted therapy for advanced RCC.

Integrin αvβ3 and fibronectin upregulate Slug in cancer cells to promote clot invasion and metastasis.

The blood clotting cascade is selectively involved in lung metastasis, but the reason for this selectivity is unclear. Here, we show that tumor cells that metastasize predominantly to the lung, such as renal cell carcinoma (RCC) and soft tissue sarcoma (STS), have an inherent capacity to generate extensive invadopodia when embedded in a blood clot. Compared with other metastatic cancer cells tested, RCC and STS cells exhibited increased levels of expression of fibronectin and an activated form of the integrin αvβ3, which coordinately supported the generation of an elaborate fibronectin matrix and actin stress fibers in fibrin-embedded tumor cells. Together, fibronectin and αvβ3 induced upregulation of the transcription factor Slug, which mediates epithelial-mesenchymal transition as well as fibrin invasion and lung metastasis. This mechanism is clinically significant, because primary cancer cells from patients with metastatic RCC strongly invaded fibrin and this correlated with fibronectin matrix formation and Slug expression. In contrast, tumor cells from patients with localized RCC were largely noninvasive. Together, our findings establish that activated integrin αvβ3 and fibronectin promote lung metastasis by upregulating Slug, defining a mechanism through which cancer cells can colonize blood clots in the lung vasculature.

NADPH Oxidase NOX4 Supports Renal Tumorigenesis by Promoting the Expression and Nuclear Accumulation of HIF2α

Most sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2α (HIF2α), a master transcriptional regulator of genes that drive diverse processes, including angiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2α expression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions of NOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cells abrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. These alterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression of manganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficient to block nuclear accumulation of HIF2α in RCC cells. Our results offer direct evidence that NOX4 is critical for renal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cells are genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade.

Hsa-miRNA-765 as a Key Mediator for Inhibiting Growth, Migration and Invasion in Fulvestrant-Treated Prostate Cancer

Fulvestrant (ICI-182,780) has recently been shown to effectively suppress prostate cancer cell growth in vitro and in vivo. But it is unclear whether microRNAs play a role in regulating oncogene expression in fulvestrant-treated prostate cancer. Here, this study reports hsa-miR-765 as the first fulvestrant-driven, ERβ-regulated miRNA exhibiting significant tumor suppressor activities like fulvestrant, against prostate cancer cell growth via blockage of cell-cycle progression at the G2/M transition, and cell migration and invasion possibly via reduction of filopodia/intense stress-fiber formation. Fulvestrant was shown to upregulate hsa-miR-765 expression through recruitment of ERβ to the 5′-regulatory-region of hsa-miR-765. HMGA1, an oncogenic protein in prostate cancer, was identified as a downstream target of hsa-miR-765 and fulvestrant in cell-based experiments and a clinical study. Both the antiestrogen and the hsa-miR-765 mimic suppressed HMGA1 protein expression. In a neo-adjuvant study, levels of hsa-miR-765 were increased and HMGA1 expression was almost completely lost in prostate cancer specimens from patients treated with a single dose (250 mg) of fulvestrant 28 days before prostatectomy. These findings reveal a novel fulvestrant signaling cascade involving ERβ-mediated transcriptional upregulation of hsa-miR-765 that suppresses HMGA1 protein expression as part of the mechanism underlying the tumor suppressor action of fulvestrant in prostate cancer.

DLK1: A Novel Target for Immunotherapeutic Remodeling of the Tumor Blood Vasculature

Tumor blood vessels are frequently inefficient in their design and function, leading to high interstitial fluid pressure, hypoxia, and acidosis in the tumor microenvironment (TME), rendering tumors refractory to the delivery of chemotherapeutic agents and immune effector cells. Here we identified the NOTCH antagonist delta-like 1 homologue (DLK1) as a vascular pericyte-associated antigen expressed in renal cell carcinomas (RCC), but not in normal kidney tissues in mice and humans. Vaccination of mice bearing established RCC against DLK1 led to immune-mediated elimination of DLK1(+) pericytes and to blood vessel normalization (i.e., decreased vascular permeability and intratumoral hypoxia) in the TME, in association with tumor growth suppression. After therapeutic vaccination, tumors displayed increased prevalence of activated VCAM1(+)CD31(+) vascular endothelial cells (VECs) and CXCL10, a type-1 T cell recruiting chemokine, in concert with increased levels of type-1 CD8(+) tumor-infiltrating lymphocytes (TIL). Vaccination against DLK1 also yielded (i) dramatic reductions in Jarid1B(+), CD133(+), and CD44(+) (hypoxia-responsive) stromal cell populations, (ii) enhanced tumor cell apoptosis, and (iii) increased NOTCH signaling in the TME. Coadministration of a γ-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl-l-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT)) that interferes with canonical NOTCH signaling resulted in the partial loss of therapeutic benefits associated with lentivirus encoding full-length murine (lvDLK1)-based vaccination.

Pharmacologic Administration of Interleukin‐2

The development of biologic therapies for patients with cancer has in part been impeded by the extraordinary complexity and intrinsic feedback mechanisms promoting homeostasis in tissue injury, repair, inflammation, and immunity. Recombinant interleukin 2 (IL‐2) therapy was initiated in 1984 based on its role as the prototypic T‐cell growth factor, with novel roles deduced late after its FDA approval in regulating not only effector T cells but also regulatory T cells. Complicating its application, even in the most sophisticated centers, has been the manageable but difficult toxicities attendant on its use in spite of clear evidence of complete responses in 5–10% of treated patients with melanoma and renal cell carcinoma with extraordinary durability lasting now for almost 25 years, thus tantamount to “cures.” Although efforts have been made to diminish toxicity or enhance efficacy the only substantive advance in combination therapy has been the application of tumor‐infiltrating lymphocytes and the antibody to CTLA4. A deeper understanding of the “limiting” toxicity associated with mild flu‐like symptoms and more debilitating cytokine “storm” not forthcoming. Here we propose the notion that the systemic syndrome associated with IL‐2 administration is due to global cytokine‐induced autophagy and temporally limited tissue dysfunction. The possible role of autophagy inhibitors to enhance efficacy and limit toxicity as well as possible problems with this approach are considered.

Inhibiting autophagy: a novel approach for the treatment of renal cell carcinoma.

AbstractThe common clear cell subtype of renal cell carcinoma is associated with hereditary or acquired loss of function of the von Hippel-Lindau tumor suppressor, a key component in oxygen sensing, perpetuating a stressed state. Autophagy is primarily a highly conserved, catabolic process by which stressed cells shuttle damaged or effete organelles and proteins into autophagosomes for sequestration and digestion after fusion with lysosomes. Autophagy is directed by autophagy-related genes and is divided into 4 discrete steps: initiation, nucleation, maturation, and degradation. During early tumorigenesis, apoptosis is enhanced and autophagy is suppressed, allowing accumulation of mutations and emergence of genomic instability. Late, an “autophagic switch” occurs, promoting survival and limiting apoptosis. Compounds such as chloroquine and hydroxychloroquine that prevent acidification of the lysosomal compartment are the sole clinically available inhibitors of autophagy. Currently, there are more than 30 trials examining combinations of hydroxychloroquine with anticancer agents. The intricate effects of autophagy on the immune response complicate manipulation of autophagy as part of the antitumor strategy. Further understanding of basic mechanisms of renal cell carcinoma pathogenesis and of autophagy will enable development of the next generation of pharmacologic modulators of autophagy.

Expression of Ror2 mediates invasive phenotypes in renal cell carcinoma

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.

Relocation of CLIC1 Promotes Tumor Cell Invasion and Colonization of Fibrin

Chloride intracellular channel 1 (CLIC1) has been shown to be upregulated in various malignancies but its exact function remains unclear. Here, it is revealed that CLIC1 is critical for the stability of invadopodia in endothelial and tumor cells embedded in a 3-dimensional (3D) matrix of fibrin. Invadopodia stability was associated with the capacity of CLIC1 to induce stress fiber and fibronectin matrix formation following its β3 integrin (ITGB3)-mediated recruitment into invadopodia. This pathway, in turn, was relevant for fibrin colonization as well as slug (SNAI2) expression and correlated with a significant role of CLIC1 in metastasis in vivo. Mechanistically, a reduction of myosin light chain kinase (MYLK) in CLIC1-depleted as well as β3 integrin-depleted cells suggests an important role of CLIC1 for integrin-mediated actomyosin dynamics in cells embedded in fibrin. Overall, these results indicate that CLIC1 is an important contributor to tumor invasion, metastasis, and angiogenesis. Implications: This study uncovers an important new function of CLIC1 in the regulation of cell–extracellular matrix interactions and ability of tumor cells to metastasize to distant organs. Mol Cancer Res; 13(2); 273–80. ©2014 AACR.