VHL loss reprograms the immune landscape to promote an inflammatory myeloid microenvironment in renal tumorigenesis.

Clear cell renal cell carcinoma (ccRCC) is characterized by dysregulated hypoxia signaling and a tumor microenvironment (TME) highly enriched in myeloid and lymphoid cells. Loss of the von Hippel Lindau (VHL) gene is a critical early event in ccRCC pathogenesis and promotes stabilization of HIF. Whether VHL loss in cancer cells affects immune cells in the TME remains unclear. Using Vhl WT and Vhl-KO in vivo murine kidney cancer Renca models, we found that Vhl-KO tumors were more infiltrated by immune cells. Tumor-associated macrophages (TAMs) from Vhl-deficient tumors demonstrated enhanced in vivo glucose consumption, phagocytosis, and inflammatory transcriptional signatures, whereas lymphocytes from Vhl-KO tumors showed reduced activation and a lower response to anti-programmed cell death 1 (anti-PD-1) therapy in vivo. The chemokine CX3CL1 was highly expressed in human ccRCC tumors and was associated with Vhl deficiency. Deletion of Cx3cl1 in cancer cells decreased myeloid cell infiltration associated with Vhl loss to provide a mechanism by which Vhl loss may have contributed to the altered immune landscape. Here, we identify cancer cell-specific genetic features that drove environmental reprogramming and shaped the tumor immune landscape, with therapeutic implications for the treatment of ccRCC.

Integrating integrins with the hallmarks of cancer.

Epithelial cells adhere to a specialized extracellular matrix called the basement membrane which allows them to polarize and form epithelial tissues. The extracellular matrix provides essential physical scaffolding and biochemical and biophysical cues required for tissue morphogenesis, differentiation, function, and homeostasis. Epithelial cell adhesion to the extracellular matrix (i.e., basement membrane) plays a critical role in organizing epithelial tissues, separating the epithelial cells from the stroma. Epithelial cell detachment from the basement membrane classically results in death, though detachment or invasion through the basement membrane represents a critical step in carcinogenesis. Epithelial cells bind to the extracellular matrix via specialized matrix receptors, including integrins. Integrins are transmembrane receptors that form a mechanical linkage between the extracellular matrix and the intracellular cytoskeleton and are required for anchorage-dependent cellular functions such as proliferation, migration, and invasion. The role of integrins in the development, growth, and dissemination of multiple types of carcinomas has been investigated by numerous methodologies, which has led to great complexity. To organize this vast array of information, we have utilized the "Hallmarks of Cancer" from Hanahan and Weinberg as a convenient framework to discuss the role of integrins in the pathogenesis of cancers. This review explores this biology and how its complexity has impacted the development of integrin-targeted anti-cancer therapeutics.

Ligand-independent integrin ß1 signaling supports lung adenocarcinoma development.

Integrins - the principal extracellular matrix (ECM) receptors of the cell - promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin ß1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin ß1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin ß1-mediated adhesion to ECM but are dependent on integrin ß1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin ß1 in lung tumorigenesis that is mediated through constitutive, ECM binding-independent signaling involving the cytoplasmic tail.

SETD2 regulates the methylation of translation elongation factor eEF1A1 in clear cell renal cell carcinoma.

BACKGROUND: SET domain-containing protein 2 (SETD2) is commonly mutated in renal cell carcinoma. SETD2 methylates histone H3 as well as a growing list of non-histone proteins. OBJECTIVE: Initially, we sought to explore SETD2-dependent changes in lysine methylation of proteins in proximal renal tubule cells. Subsequently, we focused on changes in lysine methylation of the translation elongation factor eEF1A1. METHODS: To accomplish these objectives, we initially performed a systems-wide analysis of protein lysine-methylation and expression in wild type (WT) and SETD2-knock out (KO) kidney cells and later focused our studies on eEF1A1 as well as the expression of lysine methyltransferases that regulate its lysine methylation. RESULTS: We observed decreased lysine methylation of the translation elongation factor eEF1A1. EEF1AKMT2 and EEF1AKMT3 are known to methylate eEF1A1, and we show here that their expression is dependent on SET-domain function of SETD2. Globally, we observe differential expression of hundreds of proteins in WT versus SETD2-KO cells, including increased expression of many involved in protein translation. Finally, we observe decreased progression free survival and loss of EEF1AKMT2 gene expression in SETD2-mutated tumors predicted to have loss of function of the SET domain. CONCLUSION: Overall, these data suggest that SETD2-mutated ccRCC, via loss of enzymatic function of the SET domain, displays dysregulation of protein translation as a potentially important component of the transformed phenotype.

Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages.

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.

Clinical Features and Multiplatform Molecular Analysis Assist in Understanding Patient Response to Anti-PD-1/PD-L1 in Renal Cell Carcinoma.

Predicting response to ICI therapy among patients with renal cell carcinoma (RCC) has been uniquely challenging. We analyzed patient characteristics and clinical correlates from a retrospective single-site cohort of advanced RCC patients receiving anti-PD-1/PD-L1 monotherapy (N = 97), as well as molecular parameters in a subset of patients, including multiplexed immunofluorescence (mIF), whole exome sequencing (WES), T cell receptor (TCR) sequencing, and RNA sequencing (RNA-seq). Clinical factors such as the development of immune-related adverse events (odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.05-5.91) and immunological prognostic parameters, including a higher percentage of circulating lymphocytes (23.4% vs. 17.4%, p = 0.0015) and a lower percentage of circulating neutrophils (61.8% vs. 68.5%, p = 0.0045), correlated with response. Previously identified gene expression signatures representing pathways of angiogenesis, myeloid inflammation, T effector presence, and clear cell signatures also correlated with response. High PD-L1 expression (>10% cells) as well as low TCR diversity (≤644 clonotypes) were associated with improved progression-free survival (PFS). We corroborate previously published findings and provide preliminary evidence of T cell clonality impacting the outcome of RCC patients. To further biomarker development in RCC, future studies will benefit from integrated analysis of multiple molecular platforms and prospective validation.

Neoadjuvant pazopanib and molecular analysis of tissue response in renal cell carcinoma.

BACKGROUNDSurgery remains the frontline therapy for patients with localized clear cell renal cell carcinoma (ccRCC); however, 20%-40% recur. Angiogenesis inhibitors have improved survival in metastatic patients and may result in responses in the neoadjuvant setting. The impact of these agents on the tumor genetic heterogeneity or the immune milieu is largely unknown. This phase II study was designed to evaluate safety, response, and effect on tumor tissue of neoadjuvant pazopanib.METHODSccRCC patients with localized disease received pazopanib (800 mg daily; median 8 weeks), followed by nephrectomy. Five tumors were examined for mutations by whole exome sequencing from samples collected before therapy and at nephrectomy. These samples underwent RNA sequencing; 17 samples were available for posttreatment assessment.RESULTSTwenty-one patients were enrolled. The overall response rate was 8 of 21 (38%). No patients with progressive disease. At 1-year, response-free survival and overall survival was 83% and 89%, respectively. The most frequent grade 3 toxicity was hypertension (33%, 7 of 21). Sequencing revealed strong concordance between pre- and posttreatment samples within individual tumors, suggesting tumors harbor stable core profiles. However, a reduction in private mutations followed treatment, suggesting a selective process favoring enrichment of driver mutations.CONCLUSIONNeoadjuvant pazopanib is safe and active in ccRCC. Future genomic analyses may enable the segregation of driver and passenger mutations. Furthermore, tumor infiltrating immune cells persist during therapy, suggesting that pazopanib can be combined with immune checkpoint inhibitors without dampening the immune response.FUNDINGSupport was provided by Novartis and GlaxoSmithKline as part of an investigator-initiated study.

Hypoxia-induced epithelial to mesenchymal transition in cancer.

A common feature of many solid tumors is low oxygen conditions due to inadequate blood supply. Hypoxia induces hypoxia inducible factor (HIF) stabilization and downstream signaling. This signaling has pleiotropic roles in cancers, including the promotion of cellular proliferation, changes in metabolism, and induction of angiogenesis. In addition, hypoxia is becoming recognized as an important driver of epithelial-to-mesenchymal (EMT) in cancer. During EMT, epithelial cells lose their typical polarized states and transition to a more mobile mesenchymal phenotype. Hypoxia induces this transition by modulating EMT signaling pathways, inducing EMT transcription factor activity, and regulating miRNA networks. As both hypoxia and EMT modulate the tumor microenvironment (TME) and are associated with immunosuppression, we also explore how these pathways may impact response to immuno-oncology therapeutics.

PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma.

A non-immunogenic tumor microenvironment (TME) is a significant barrier to immune checkpoint blockade (ICB) response. The impact of Polybromo-1 (PBRM1) on TME and response to ICB in renal cell carcinoma (RCC) remains to be resolved. Here we show that PBRM1/Pbrm1 deficiency reduces the binding of brahma-related gene 1 (BRG1) to the IFNγ receptor 2 (Ifngr2) promoter, decreasing STAT1 phosphorylation and the subsequent expression of IFNγ target genes. An analysis of 3 independent patient cohorts and of murine pre-clinical models reveals that PBRM1 loss is associated with a less immunogenic TME and upregulated angiogenesis. Pbrm1 deficient Renca subcutaneous tumors in mice are more resistance to ICB, and a retrospective analysis of the IMmotion150 RCC study also suggests that PBRM1 mutation reduces benefit from ICB. Our study sheds light on the influence of PBRM1 mutations on IFNγ-STAT1 signaling and TME, and can inform additional preclinical and clinical studies in RCC.

ß1 Integrin regulates adult lung alveolar epithelial cell inflammation.

Integrins, the extracellular matrix receptors that facilitate cell adhesion and migration, are necessary for organ morphogenesis; however, their role in maintaining adult tissue homeostasis is poorly understood. To define the functional importance of ß1 integrin in adult mouse lung, we deleted it after completion of development in type 2 alveolar epithelial cells (AECs). Aged ß1 integrin-deficient mice exhibited chronic obstructive pulmonary disease-like (COPD-like) pathology characterized by emphysema, lymphoid aggregates, and increased macrophage infiltration. These histopathological abnormalities were preceded by ß1 integrin-deficient AEC dysfunction such as excessive ROS production and upregulation of NF-κB-dependent chemokines, including CCL2. Genetic deletion of the CCL2 receptor, Ccr2, in mice with ß1 integrin-deficient type 2 AECs impaired recruitment of monocyte-derived macrophages and resulted in accelerated inflammation and severe premature emphysematous destruction. The lungs exhibited reduced AEC efferocytosis and excessive numbers of inflamed type 2 AECs, demonstrating the requirement for recruited monocytes/macrophages in limiting lung injury and remodeling in the setting of a chronically inflamed epithelium. These studies support a critical role for ß1 integrin in alveolar homeostasis in the adult lung.

Strategies to overcome therapeutic resistance in renal cell carcinoma.

BACKGROUND: Renal cell cancer (RCC) is a prevalent and lethal disease. At time of diagnosis, most patients present with localized disease. For these patients, the standard of care includes nephrectomy with close monitoring thereafter. While many patients will be cured, 5-year recurrence rates range from 30% to 60%. Furthermore, nearly one-third of patients present with metastatic disease at time of diagnosis. Metastatic disease is rarely curable and typically lethal. Cytotoxic chemotherapy and radiation alone are incapable of controlling the disease. Extensive effort was expended in the development of cytokine therapies but response rates remain low. Newer agents targeting angiogenesis and mTOR signaling emerged in the 2000s and revolutionized patient care. While these agents improve progression free survival, the development of resistance is nearly universal. A new era of immunotherapy is now emerging, led by the checkpoint inhibitors. However, therapeutic resistance remains a complex issue that is likely to persist. METHODS AND PURPOSE: In this review, we systematically evaluate preclinical research and clinical trials that address resistance to the primary RCC therapies, including anti-angiogenesis agents, mTOR inhibitors, and immunotherapies. As clear cell RCC is the most common adult kidney cancer and has been the focus of most studies, it will be the focus of this review.

Renal cancer subtypes: Should we be lumping or splitting for therapeutic decision making?

The treatment of advanced renal cell carcinoma has posed a challenge for decades, in part because of common themes related to intrinsic resistance to cytotoxic chemotherapy and the obscure biology of these cancer types. Forward movement in the treatment of the renal cell carcinomas thus can be approached in 2 ways: by splitting the tumor types along histologic and molecular features, in the hopes of coupling highly precision-focused therapy on a subset of patients who have disease with the most potential for benefit; or by lumping the various biologies and histologies together, to include the rarer renal cell carcinoma types with the more common types. The former strategy satisfies the desire for customized precision in treatment delivery, whereas the latter strategy allows clinicians to offer a wider therapeutic menu in a set of diseases we are continuing to learn about on a physiologic and molecular level. Cancer 2017;123:200-209. © 2016 American Cancer Society.

Insights into the Genetic Basis of the Renal Cell Carcinomas from The Cancer Genome Atlas.

The renal cell carcinomas (RCC), clear cell, papillary, and chromophobe, have recently undergone an unmatched genomic characterization by The Cancer Genome Atlas. This analysis has revealed new insights into each of these malignancies and underscores the unique biology of clear cell, papillary, and chromophobe RCC. Themes that have emerged include distinct mechanisms of metabolic dysregulation and common mutations in chromatin modifier genes. Importantly, the papillary RCC classification encompasses a heterogeneous group of diseases, each with highly distinct genetic and molecular features. In conclusion, this review summarizes RCCs that represent a diverse set of malignancies, each with novel biologic programs that define new paradigms for cancer biology. Mol Cancer Res; 14(7); 589-98. ©2016 AACR.

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics.

PURPOSE: Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. EXPERIMENTAL DESIGN: Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. To describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. RESULTS: Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, whereas DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. CONCLUSIONS: Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology among kidney cancer subtypes. Clin Cancer Res; 22(22); 5605-16. ©2016 AACR.

Patients with ClearCode34-identified molecular subtypes of clear cell renal cell carcinoma represent unique populations with distinct comorbidities.

PURPOSE: The 34-gene classifier, ClearCode34, identifies prognostically distinct molecular subtypes of clear cell renal cell carcinoma (ccRCC) termed clear cell A (ccA) and clear cell B (ccB). The primary objective of this study was to describe clinical characteristics and comorbidities of relevance in patients stratified by ClearCode34. PATIENTS AND METHODS: In this retrospective analysis, 282 patients from Moffitt Cancer Center with ccRCC with gene expression analyses of the primary tumor were identified and ClearCode34 was applied to identify tumors as ccA or ccB. The medical record and institutional databases were queried to define patient characteristics, comorbidities, and outcomes. RESULTS: We validated in this external cohort the superior overall survival, cancer-specific survival, and recurrence-free survival of ccA patients relative to ccB patients (P<0.001). Addressing other clinical characteristics, the ccA patients were more likely to be obese (48% vs. 34%, P = 0.021) and diabetic (26% vs. 13%, P = 0.035). The ccA patients also trended toward having been more frequent users of angiotensin system inhibitors (71% vs. 52%, P = 0.055). In multivariate analyses, ccB status is independently associated with inferior cancer-specific survival (hazard ratio = 3.26, 95% confidence interval: 1.84-5.79) and overall survival (hazard ratio = 2.50, 95% confidence interval: 1.53-4.08). CONCLUSIONS: ClearCode34, after considering distinct patterns of comorbidities in each molecular subtype, remains a strong prognostic tool in patients with ccRCC. Obesity and diabetes mellitus emerged as factors that may influence ccRCC phenotypes and further studies investigating the effect of these metabolic conditions functionally onto tumor biology are warranted. Additionally, use of angiotensin system inhibitors could be studied in the context of ccRCC molecular classification in future studies to better understand its effect on ccRCC outcomes.

Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays.

Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. We used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations, and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules.

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.

Conservation of hearing and protection of auditory hair cells against trauma-induced losses by local dexamethasone therapy: molecular and genetic mechanisms.

HYPOTHESIS: Dexamethasone (DXM) protects hearing against trauma-induced loss. MATERIALS: in vivo: A guinea pig model of electrode induced trauma (EIT)-induced hearing loss was used to locally deliver dexamethasone. In vitro: TNF-α-challenged organ of Corti explants treated with DXM or polymer-eluted DXM +/- PI3K/Akt/PkB/NFkB inhibitors were used for hair cells count and gene expression studies. RESULTS: in vivo: local DXM treatment of EIT-animals prevents trauma-induced loss of ABR thresholds that occurs in EIT-animals and EIT-animals treated with the carrier solution (i.e., AP), and prevented loss of auditory hair cells. In vitro: DXM and polymer-eluted DXM were equally effective in protecting hair cells from ototoxic levels of TNF-α Inhibitor treated explants demonstrated that DXM treatment requires both Akt/PKB and NFkB signalling for otoprotection. DXM treatment of explants showed up regulation of anti-apoptosis related genes (i.e., Bcl-2, Bcl-xl) and down regulation of pro-apoptosis related genes (i.e., Bax, TNFR-1). CONCLUSIONS: DXM exert its otoprotective action by activation of cell signal molecules (e.g., NFkB) that alter the expression of anti- and pro-apoptosis genes.

Dexamethasone protects auditory hair cells against TNFalpha-initiated apoptosis via activation of PI3K/Akt and NFkappaB signaling.

BACKGROUND: Tumor necrosis factor alpha (TNFalpha) is associated with trauma-induced hearing loss. Local treatment of cochleae of trauma-exposed animals with a glucocorticoid is effective in reducing the level of hearing loss that occurs post-trauma (e.g., electrode insertion trauma-induced hearing loss/dexamethasone treatment). HYPOTHESIS: Dexamethasone (Dex) protects auditory hair cells (AHCs) from trauma-induced loss by activating cellular signal pathways that promote cell survival. MATERIALS AND METHODS: Organ of Corti explants challenged with an ototoxic level of TNFalpha was the trauma model with Dex the otoprotective drug. A series of inhibitors were used in combination with the Dex treatment of TNFalpha-exposed explants to investigate the signal molecules that participate in Dex-mediated otoprotection. The otoprotective capacity of Dex against TNFalpha ototoxicity was determined by hair cell counts obtained from fixed explants stained with FITC-phalloidin labeling with investigators blinded to specimen identity. RESULTS: The general caspase inhibitor Boc-d-fmk prevented TNFalpha-induced AHC death. There was a significant reduction (p<0.05) in the efficacy of Dex otoprotection against TNFalpha ototoxicity when the following cellular events were blocked: (1) glucocorticoid receptors (Mif); (2) PI3K (LY294002); (3) Akt/PKB (SH-6); and (4) NFkappaB (NFkappaB-I). CONCLUSION: Dex treatment protects hair cells against TNFalpha apoptosis in vitro by activation of PI3K/Akt and NFkappaB signaling.