John D. Gordan

The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase

ABSTRACT The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.

HIF-α Effects on c-Myc Distinguish Two Subtypes of Sporadic VHL-Deficient Clear Cell Renal Carcinoma

von Hippel-Lindau (VHL) tumor suppressor loss results in hypoxia-inducible factor alpha (HIF-alpha) stabilization and occurs in 70% of sporadic clear cell renal carcinomas (ccRCCs). To determine whether opposing influences of HIF-1alpha and HIF-2alpha on c-Myc activity regulate human ccRCC progression, we analyzed VHL genotype and HIF-alpha expression in 160 primary tumors, which segregated into three groups with distinct molecular characteristics. Interestingly, ccRCCs with intact VHL, as well as pVHL-deficient HIF-1alpha/HIF-2alpha-expressing ccRCCs, exhibited enhanced Akt/mTOR and ERK/MAPK signaling. In contrast, pVHL-deficient ccRCCs expressing only HIF-2alpha displayed elevated c-Myc activity, resulting in enhanced proliferation and resistance to replication stress. These reproducible distinctions in ccRCC behavior delineate HIF-alpha effects on c-Myc in vivo and suggest molecular criteria for selecting targeted therapies.

HIF2α inhibition promotes p53 pathway activity, tumor cell death, and radiation responses

Approximately 50% of cancer patients receive radiation treatment, either alone or in combination with other therapies. Tumor hypoxia has long been associated with resistance to radiation therapy. Moreover, the expression of hypoxia inducible factors HIF1α and/or HIF2α correlates with poor prognosis in many tumors. Recent evidence indicates that HIF1α expression can enhance radiation-induced apoptosis in cancer cells. We demonstrate here that HIF2α inhibition promotes tumor cell death and, in contrast to HIF1α, enhances the response to radiation treatment. Specifically, inhibiting HIF2α expression augments p53 activity, increases apoptosis, and reduces clonogenic survival of irradiated and non-irradiated cells. Moreover, HIF2α inhibition promotes p53-mediated responses by disrupting cellular redox homeostasis, thereby permitting reactive oxygen species (ROS) accumulation and DNA damage. These results correlate with altered p53 phosphorylation and target gene expression in untreated human tumor samples and show that HIF2α likely contributes to tumor cell survival including during radiation therapy.

Hypoxia-mediated Selective mRNA Translation by an Internal Ribosome Entry Site-independent Mechanism

Although it is advantageous for hypoxic cells to inhibit protein synthesis and conserve energy, it is also important to translate mRNAs critical for adaptive responses to hypoxic stress. Because internal ribosome entry sites (IRES) have been postulated to mediate this preferential synthesis, we analyzed the 5 ′-untranslated regions from a panel of stress-regulated mRNAs for m7GTP cap-independent translation and identified putative IRES elements in encephalomyocarditis virus, vascular endothelial growth factor, hypoxia-inducible factors (HIFs) 1α and 2α, glucose transporter-like protein 1, p57Kip2, La, BiP, and triose phosphate isomerase transcripts. However, when capped and polyadenylated dicistronic RNAs were synthesized in vitro and transfected into cells, cellular IRES-mediated translation accounted for less than 1% that of the level of cap-dependent translation. Moreover, hypoxic stress failed to activate cap-independent synthesis, indicating that it is unlikely that this is the primary mechanism for the maintenance of the translation of these mRNAs under low O2. Furthermore, although HIF-1α is frequently cited as an example of an mRNA that is preferentially translated, we demonstrate that under different levels and durations of hypoxic stress, changes in newly synthesized HIF-1α and β-actin protein levels mirror alterations in corresponding mRNA abundance. In addition, our data suggest that cyclin-dependent kinase inhibitor p57Kip2 and vascular endothelial growth factor mRNAs are selectively translated by an IRES-independent mechanism under hypoxic stress.

Ribosomal Stress Couples the Unfolded Protein Response to p53-dependent Cell Cycle Arrest

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2α leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK-/- murine embryonic fibroblasts have an attenuated G1/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

ADAP–SLP-76 Binding Differentially Regulates Supramolecular Activation Cluster (SMAC) Formation Relative to T Cell–APC Conjugation

T cell–APC conjugation as mediated by leukocyte function-associated antigen-1 (LFA-1)–intercellular adhesion molecule (ICAM)-1 binding is followed by formation of the supramolecular activation cluster (SMAC) at the immunological synapse. The intracellular processes that regulate SMAC formation and its influence on T cell function are important questions to be addressed. Here, using a mutational approach, we demonstrate that binding of adaptor adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation relative to conjugation. Although mutation of the YDDV sites (termed M12) disrupted SLP-76 SH2 domain binding and prevented the ability of ADAP to increase conjugation and LFA-1 clustering, M12 acted selectively as a dominant negative (DN) inhibitor of pSMAC formation, an effect that was paralleled by a DN effect on interleukin-2 production. ADAP also colocalized with LFA-1 at the immunological synapse. Our findings identify ADAP–SLP-76 binding as a signaling event that differentially regulates SMAC formation, and support a role for SMAC formation in T cell cytokine production.

Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma

Inappropriate kinase expression and subsequent promiscuous activity defines the transformation of many solid tumors including renal cell carcinoma (RCC). Thus, the expression of novel tumor-associated kinases has the potential to dramatically shape tumor cell behavior. Further, identifying tumor-associated kinases can lend insight into patterns of tumor growth and characteristics. Here, we report the identification of the RTK-like orphan receptor 2 (Ror2), a new tumor-associated kinase in RCC cell lines and primary tumors. Ror2 is an orphan receptor tyrosine kinase with physiological expression normally seen in the embryonic kidney. However, in RCC, Ror2 expression correlated with expression of genes involved at the extracellular matrix, including Twist and matrix metalloprotease-2 (MMP2). Expression of MMP2 in RCC cells was suppressed by Ror2 knockdown, placing Ror2 as a mediator of MMP2 regulation in RCC and a potential regulator of extracellular matrix remodeling. The suppression of Ror2 not only inhibited cell migration, but also inhibited anchorage-independent growth in soft agar and growth in an orthotopic xenograft model. These findings suggest a novel pathway of tumor-promoting activity by Ror2 within a subset of renal carcinomas, with significant implications for unraveling the tumorigenesis of RCC.

Integrative Genomic Analyses of Sporadic Clear Cell Renal Cell Carcinoma Define Disease Subtypes and Potential New Therapeutic Targets

Sporadic clear cell renal cell carcinoma (ccRCC), the most common type of adult kidney cancer, is often associated with genomic copy number aberrations on chromosomes 3p and 5q. Aberrations on chromosome 3p are associated with inactivation of the tumor suppressor gene von-Hippel Lindau (VHL), which activates the hypoxia-inducible factors HIF1α and HIF2α. In contrast, ccRCC genes on chromosome 5q remain to be defined. In this study, we conducted an integrated analysis of high-density copy number and gene expression data for 54 sporadic ccRCC tumors that identified the secreted glycoprotein STC2 (stanniocalcin 2) and the proteoglycan VCAN (versican) as potential 5q oncogenes in ccRCCs. In functional assays, STC2 and VCAN each promoted tumorigenesis by inhibiting cell death. Using the same approach, we also investigated the two VHL-deficient subtypes of ccRCC, which express both HIF1α and HIF2α (H1H2) or only HIF2α (H2). This analysis revealed a distinct pattern of genomic aberrations in each group, with the H1H2 group displaying, on average, a more aberrant genome than the H2 group. Together our findings provide a significant advance in understanding ccRCCs by offering a molecular definition of two subtypes with distinct characteristics as well as two potential chromosome 5q oncogenes, the overexpression of which is sufficient to promote tumorigenesis by limiting cell death.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1–S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472–84. ©2016 AACR.

Linking Tumor Mutations to Drug Responses via a Quantitative Chemical–Genetic Interaction Map

UNLABELLED There is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic-lethal genetic interactions or dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated a systematic and quantitative chemical-genetic interaction map that charts the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model complex cellular contexts. Applying this dataset to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene, including resistance to AKT-PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can accelerate the development of new genotype-directed therapies. SIGNIFICANCE Determining how the plethora of genomic abnormalities that exist within a given tumor cell affects drug responses remains a major challenge in oncology. Here, we develop a new mapping approach to connect cancer genotypes to drug responses using engineered isogenic cell lines and demonstrate how the resulting dataset can guide clinical interrogation.