Search

Understanding the physiology and functions of the liver and cancer requires knowing transcriptional patterns driving biological activities within the functional structures of the tissue, especially the zonated features of the liver metabolic networks. Using the powerful and unique capabilities of GeoMx® Digital Spatial Profiler (DSP) with the Whole Transcriptome Atlas (WTA) panel to resolve functional units within FFPE tissues in situ, here we report the spatial analysis of whole transcriptomes across three micro-dissected zones (pericentral zone 3, intermediate zone 2 and periportal zone 1) of human normal liver and HCC. We also report the whole transcriptome expression data from Kupffer cells, portal traits and interlobular bile ducts from four normal liver samples and HCC. 500 - 3000 genes were detected from functional groups within each histological structure. In the liver functional units, 1000 - 2500 genes were detected in zone 1, 2 and 3 separately. By comparing the whole transcriptome profiles of zone 1 and zone 3, we have found 32 differentially expressed targets (fold change > 1.5, p value < 0.05) which showed a gradient expression pattern along the porto-central axis. The expression patterns of CYP1A2, CYP2E1, CYP3A4 and ALDOB matched well with their respective patterns of protein expression (Human Protein Atlas), recapitulating the well-studied distribution of functional activities along the porto-central axis. Moreover, by combining with Gene Set Enrichment Analysis (GSEA), we have found important pathways involved in metabolisms in either the pericentral area or the periportal area. Pathways including biological oxidations (CYP1A2, CYP2E1, CYP3A4, ADH1A, and ADH1B) and lipids metabolism (AKR1C1, AKR1C2, and SLCO1B3) showed high enrichment in zone 3 and decreased towards zone 1. In contrast, pathways including platelet degranulation (FGA, FGB, FGG), glucose metabolism (ALDOB and PCK1) and amino acids metabolism (HAL, SDS, NNMT, and GLS2) showed high enrichment in zone 1 and decreased towards zone 3. In conclusion, our WTA data has revealed clear metabolic zonation in the liver along the porto-central axis. GeoMx technology with WTA is a powerful tool to investigate the underlying mechanisms of liver metabolism, regeneration, and tissue structure. It can be further utilized to study the whole transcriptomic differences in normal and diseased tissue. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Yan Liang, Nan Wang, Xia Li, Kathy Ton, Liang Zhang, Megan Vandenberg, Charlie Glaser, Zhiyong Ding, Joseph Beechem, Andy Nam, Yan Liang. Spatial whole transcriptome profiling of human normal liver and HCC uncovers unique insights into metabolic zonation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4709.

Alterations in DNAs could not reveal what happened in proteins. The accumulated alterations of DNAs would change the manifestation of proteins. Therefore, as is the case in cancer liquid biopsies, deep proteome profiling will likely provide invaluable and clinically relevant information in real-time throughout all stages of cancer progression. However, due to the great complexity of proteomes in liquid biopsy samples and the limitations of proteomic technologies compared to high-plex sequencing technologies, proteomic discoveries have yet lagged behind their counterpart, genomic technologies. Therefore, novel protein technologies are in urgent demand to fulfill the goals set out for biomarker discovery in cancer liquid biopsies.Notably, conventional and innovative technologies are being rapidly developed for proteomic analysis in cancer liquid biopsies. These advances have greatly facilitated early detection, diagnosis, prognosis, and monitoring of cancer evolution, adapted or adopted in response to therapeutic interventions. In this paper, we review the high-plex proteomics technologies that are capable of measuring at least hundreds of proteins simultaneously from liquid biopsy samples, ranging from traditional technologies based on mass spectrometry (MS) and antibody/antigen arrays to innovative technologies based on aptamer, proximity extension assay (PEA), and reverse phase protein arrays (RPPA).

Purpose: B-cell lymphoma-2 (BCL-2), an antiapoptotic protein often dysregulated in B-cell lymphomas, promotes cell survival and provides protection from stress. A recent phase I first-in-human study of the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma showed an overall response rate of 44%. These promising clinical results prompted our examination of the biological effects and mechanism of action underlying venetoclax activity in aggressive B-cell lymphoma, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Experimental Design: MCL and DLBCL cell lines, primary patient samples, and in vivo patient-derived xenograft (PDX) models were utilized to examine venetoclax efficacy. Furthermore, the mechanisms underlying venetoclax response and the development of venetoclax resistance were evaluated using proteomics analysis and Western blotting. Results: Potential biomarkers linked to venetoclax activity and targeted combination therapies that can augment venetoclax response were identified. We demonstrate that DLBCL and MCL cell lines, primary patient samples, and PDX mouse models expressing high BCL-2 levels are extremely sensitive to venetoclax treatment. Proteomics studies showed that venetoclax substantially alters the expression levels and phosphorylation status of key proteins involved in cellular processes, including the DNA damage response, cell metabolism, cell growth/survival, and apoptosis. Short- and long-term exposure to venetoclax inhibited PTEN expression, leading to enhanced AKT pathway activation and concomitant susceptibility to PI3K/AKT inhibition. Intrinsic venetoclax-resistant cells possess high AKT activation and are highly sensitive to PI3K/AKT inhibition. Conclusions: These findings demonstrate the on-target effect of venetoclax and offer potential mechanisms to overcome acquired and intrinsic venetoclax resistance through PI3K/AKT inhibition. Clin Cancer Res; 24(16); 3967–80. ©2018 AACR.

Background Extracellular matrix (ECM) is a mediator of tumor progression. However, whether the alterations of the intraperitoneal ECM prior to tumor establishment affects the malignant progression of ovarian cancer remains elusive. Methods Apolipoprotein (ApoE) knock-out mice was used to analyze the intraperitoneal ECM alterations by quantification of the major components of ECM. ID8 cells were implanted in vivo to generate allografts and human ovarian cancer cell lines were characterized in vitro to assess the effects of ECM alterations on the malignant progression of ovarian cancer. Adhesion assay, immunochemistry, cytokines profile, proliferation assay, transwell invasion assay and western blot were used to determine the malignant phenotype of ovarian cancer cells. Results ApoE loss induced increased ECM deposition, which stimulated the adhesions of ovarian cancer cells. The adhesion-mediated focal adhesion kinase (FAK) signaling enhanced the invasive behaviors of ovarian cancer cells through activation of a ERK-MMP linkage. This ECM-induced signaling cascade was further confirmed in human ovarian cancer cell lines in vitro. Furthermore, reversal of the ECM accumulation with BAPN or abrogation of adhesion-induced ERK activation in ovarian cancer cells with MEK inhibitors (MEKi) was found to effectively delay ovarian cancer progression. Conclusions These findings identify the FAK-ERK activation in cell/matrix adhesion in the malignant progression of ovarian cancer and the efficiency of BAPN or MEKi for tumor suppression, providing an impetus for further studies to explore the possibility of new anticancer therapeutic combinations. Electronic supplementary material The online version of this article (10.1186/s13046-018-0696-4) contains supplementary material, which is available to authorized users.

Chromophobe renal cell carcinoma (ChRCC) is characterized by major changes in chromosomal copy number (CN). No model is available to precisely elucidate the molecular drivers of this tumor type. HNF1B is a master regulator of gene expression. Here, we report that the transcription factor HNF1B is downregulated in the majority of ChRCC and that the magnitude of HNF1B loss is unique to ChRCC. We also observed a strong correlation between reduced HNF1B expression and aneuploidy in ChRCC patients. In murine embryonic fibroblasts or ACHN cells, HNF1B deficiency reduced expression of the spindle checkpoint proteins MAD2L1 and BUB1B, and the cell-cycle checkpoint proteins RB1 and p27. Furthermore, it altered the chromatin accessibility of Mad2l1, Bub1b, and Rb1 genes and triggered aneuploidy development. Analysis of The Cancer Genome Atlas database revealed TP53 mutations in 33% of ChRCC where HNF1B expression was repressed. In clinical specimens, combining HNF1B loss with TP53 mutation produced an association with poor patient prognosis. In cells, combining HNF1B loss and TP53 mutation increased cell proliferation and aneuploidy. Our results show how HNF1B loss leads to abnormal mitotic protein regulation and induction of aneuploidy. We propose that coordinate loss of HNF1B and TP53 may enhance cellular survival and confer an aggressive phenotype in ChRCC. Cancer Res; 77(19); 5313–26. ©2017 AACR.

We demonstrate that concurrent administration of poly(ADP-ribose) polymerase (PARP) and WEE1 inhibitors is effective in inhibiting tumor growth but poorly tolerated. Concurrent treatment with PARP and WEE1 inhibitors induces replication stress, DNA damage, and abrogates the G2 DNA damage checkpoint in both normal and malignant cells. Following cessation of monotherapy with PARP or WEE1 inhibitors, effects of these inhibitors persist suggesting that sequential administration of PARP and WEE1 inhibitors could maintain efficacy while ameliorating toxicity. Strikingly, while sequential administration mirrored concurrent therapy in cancer cells that have high basal replication stress, low basal replication stress in normal cells protected them from DNA damage and toxicity, thus improving tolerability while preserving efficacy in ovarian cancer xenograft and patient-derived xenograft models.

Extracellular matrix (ECM) is a mediator of tumor progression. However, whether the alterations of the intraperitoneal ECM prior to tumor establishment affects the malignant progression of ovarian cancer remains elusive.Apolipoprotein (ApoE) knock-out mice was used to analyze the intraperitoneal ECM alterations by quantification of the major components of ECM. ID8 cells were implanted in vivo to generate allografts and human ovarian cancer cell lines were characterized in vitro to assess the effects of ECM alterations on the malignant progression of ovarian cancer. Adhesion assay, immunochemistry, cytokines profile, proliferation assay, transwell invasion assay and western blot were used to determine the malignant phenotype of ovarian cancer cells.ApoE loss induced increased ECM deposition, which stimulated the adhesions of ovarian cancer cells. The adhesion-mediated focal adhesion kinase (FAK) signaling enhanced the invasive behaviors of ovarian cancer cells through activation of a ERK-MMP linkage. This ECM-induced signaling cascade was further confirmed in human ovarian cancer cell lines in vitro. Furthermore, reversal of the ECM accumulation with BAPN or abrogation of adhesion-induced ERK activation in ovarian cancer cells with MEK inhibitors (MEKi) was found to effectively delay ovarian cancer progression.These findings identify the FAK-ERK activation in cell/matrix adhesion in the malignant progression of ovarian cancer and the efficiency of BAPN or MEKi for tumor suppression, providing an impetus for further studies to explore the possibility of new anticancer therapeutic combinations.

Background In 2016, it is estimated that there will be 62,700 new cases of kidney cancer in the United States, and 14,240 patients will die from the disease. Because the incidence of kidney renal clear cell carcinoma (KIRC), the most common type of kidney cancer, is expected to continue to increase in the US, there is an urgent need to find effective diagnostic biomarkers for KIRC that could help earlier detection of and customized treatment strategies for the disease. Accordingly, in this study we systematically investigated KIRC’s prognostic biomarkers for survival using the reverse phase protein array (RPPA) data and the high throughput sequencing data from The Cancer Genome Atlas (TCGA). Results With comprehensive data available in TCGA, we systematically screened protein expression based survival biomarkers in 10 major cancer types, among which KIRC presented many protein prognostic biomarkers of survival time. This is in agreement with a previous report that expression level changes (mRNAs, microRNA and protein) may have a better performance for prognosis of KIRC. In this study, we also identified 52 prognostic genes for KIRC, many of which are involved in cell-cycle and cancer signaling, as well as 15 tumor-stage-specific prognostic biomarkers. Notably, we found fewer prognostic biomarkers for early-stage than for late-stage KIRC. Four biomarkers (the RPPA protein IDs: FASN, ACC1, Cyclin_B1 and Rad51) were found to be prognostic for survival based on both protein and mRNA expression data. Conclusions Through pan-cancer screening, we found that many protein biomarkers were prognostic for patients’ survival in KIRC. Stage-specific survival biomarkers in KIRC were also identified. Our study indicated that these protein biomarkers might have potential clinical value in terms of predicting survival in KIRC patients and developing individualized treatment strategies. Importantly, we found many biomarkers in KIRC at both the mRNA expression level and the protein expression level. These biomarkers shared a significant overlap, indicating that they were technically replicable. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4026-6) contains supplementary material, which is available to authorized users.

Cyclin E is altered in nearly a third of invasive breast cancers where it is a powerful independent predictor of survival in women with stage I–III disease. Full-length cyclin E is posttranslationally cleaved into low molecular weight (LMW-E) isoforms, which are tumor-specific and accumulate in the cytoplasm because they lack a nuclear localization sequence. We hypothesized that aberrant localization of cytosolic LMW-E isoforms alters target binding and activation ultimately contributing to LMW-E–induced tumorigenicity. To address this hypothesis, we used a retrovirus-based protein complementation assay to find LMW-E binding proteins in breast cancer, identifying ATP-citrate lyase (ACLY), an enzyme in the de novo lipogenesis pathway, as a novel LMW-E–interacting protein in the cytoplasm. LMW-E upregulated ACLY enzymatic activity, subsequently increasing lipid droplet formation, thereby providing cells with essential building blocks to support growth. ACLY was also required for LMW-E–mediated transformation, migration, and invasion of breast cancer cells in vitro along with tumor growth in vivo. In clinical specimens of breast cancer, the absence of LMW-E and low expression of adipophilin (PLIN2), a marker of lipid droplet formation, associated with favorable prognosis, whereas overexpression of both proteins correlated with a markedly worse prognosis. Taken together, our findings establish a novel relationship between LMW-E isoforms of cyclin E and aberrant lipid metabolism pathways in breast cancer tumorigenesis, warranting further investigation in additional malignancies exhibiting their expression. Cancer Res; 76(8); 2406–18. ©2016 AACR.

Background Chemo-resistance is still a major obstacle in efforts to overcome acute myeloid leukemia (AML). An emerging concept has proposed that interactions between the bone marrow (BM) microenvironment and leukemia cells reduce the sensitivity of the leukemia cells to chemotherapy. As an important element of the tumor microenvironment, the cancer-associated fibroblasts (CAFs) are considered to be activated modulators in the chemo-resistance of many solid tumors. But their contribution to AML has yet to be fully understood. Here we report a critical role for CAFs which were thought to be a survival and chemo-protective factor for leukemia cells. Methods A retrospective study on the BM biopsies from 63 primary AML patients and 59 normal controls was applied to quantitative analysis the fiber stroma in the BM sections. Then immunohistochemistry on the BM biopsies were used to detect the makers of the CAFs. Their effects on drug resistance of leukemia cells were further to be assessed by co-cultured experiments in vitro. Moreover, the possible mechanisms involved in CAF-mediated chemo-protection of AML cells was investigated by antibody neutralization and siRNA knockdown experiments, with particular emphasis on the role of GDF15. Results In our study, excessive reticular fibers in the BM led to higher frequency of relapse and mortality in primary AML patients, bringing the inspiration for us to investigate the functional roles of the fiber-devied cells. We declared that the CAF cells which expressed higher levels of FSP1, α-SMA or FAP protein were widely distributed in the marrow of AML. Then in vitro co-cultured tests showed that these CAFs could protect leukemia cell lines (THP-1/K562) from chemotherapy. Interestingly, this effect could be decreased by either treatment with a neutralizing anti-GDF15 antibody or knockdown GDF15 (with siGDF15) in CAFs. Furthermore, we also confirmed that the GDF15+ cells mainly co-localized with FAP, which was identified as the typical phenotype of CAFs in the BM stroma. Conclusions We firstly demonstrate that the functional CAFs are widespread within the BM of AML patients and should be a critical chemo-protective element for AML cells by producing amount of GDF15. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0405-0) contains supplementary material, which is available to authorized users.

Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival.

Aberrant von Hippel Lindau (VHL) protein function is the underlying driver of VHL-related diseases, including both sporadic and inherited clear cell renal cell carcinoma (ccRCC). About one third of VHL mutations are missense point mutations, with R167Q being the most common VHL point mutation in hereditary VHL disease. Although it has been studied extensively, the ability of VHL-R167Q to downregulate hypoxia-inducible factor 2α (HIF2α) is still controversial. In addition, the manner in which the mutation contributes to tumorigenesis is not fully understood. No therapeutic approach is available to target VHL-R167Q and similar missense point mutations. We analyzed VHL-R167Q proteostasis and function at normoxia, at hypoxia with different oxygen pressure, and in a xenograft mouse model. We showed that the protein levels of VHL-R167Q dictate its ability to downregulate HIF2α and suppress tumor growth. Strikingly, the proteasome inhibitors bortezomib and carfilzomib, which are currently in clinical use, stabilize VHL-R167Q and increase its ability to downregulate HIF2α. VHL-R167Q binds elongin C and elongin B with considerably less avidity than wild-type VHL does but retains residual capacity to generate a VHL-elongin C-elongin B complex, downregulate HIF2α, and suppress tumorigenesis, which could be rescued by increase of VHL-R167Q levels. Finally, we used in silico approaches and identified other missense VHL mutants in addition to VHL-R167Q that might be rescued by similar strategies. Thus, our studies revealed detailed information describing how VHL-R167Q contributes to tumorigenesis and identified a potential targeted therapy for ccRCC and other VHL-related disease in patients carrying VHL-R167Q or similar missense mutations. Cancer Res; 74(11); 3127–36. ©2014 AACR.

614 Background: Recently we reported that antiangiogenic therapy-treated primary RCC demonstrated increased infiltration of CD4 and CD8 T cells and higher expression of the immune checkpoint ligand PD-L1, all of which were associated with therapy resistance. IFNg-STAT1 signaling pathway drives the expression of both immunosuppressive and immunostimulatory molecules that are involved in lymphocyte trafficking, antigen presentation and immune checkpoints. The purpose of this study was to assess the impact of antiangiogenic therapy on IFNg signaling and on a broader tumor specific immune profile. Methods: We performed immunohistochemical staining of tissue microarrays (TMAs) generated from 40 sunitinib treated primary RCC from patients treated on clinical trial and compared stains to untreated control tumors. We used Vectra imaging to spectrally unmix antibody signal and to quantitate expression levels. We used immunoblot and real-time PCR to investigate the changes in protein and mRNA, respectively. We assessed for statistical significance using an unpaired Student t test. Results: We found that sunitinib treated RCC tissues demonstrated STAT1 phosphorylation (1.2% vs. 4.8%; P

The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type–specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention.

Metabolic stress, such as an insufficient supply of nutrients or oxygen owing to inadequate tumor neovascularization, metastasis, or therapy, is a microenvironmental condition frequently encountered by cancer cells [1]. The ability to survive metabolic stress is thus a necessity for tumor initiation and progression. The serine/threonine kinase AKT (also known as protein kinase B) is a primary mediator of survival of both normal and malignant cells [2]. Our recent studies revealed that AKT could be differentially activated by site-specific phosphorylation according to the severity and duration of metabolic stress, enabling cells to shift gears to survive [3]. During canonical AKT activation, growth factors or other stimuli activate transmembrane receptor tyrosine kinases, which in turn activate phosphoinositide 3-kinase to phosphorylate phosphatidylinositol 4, 5-bisphosphate to form phosphatidylinositol 3, 4, 5-trisphosphate on the inner cell membrane. AKT and its upstream kinase, phosphoinositide-dependent kinase-1 (PDK1), are recruited to the cell membrane, which initiates AKT phosphorylation at Thr308 by PDK1 [4]. Mammalian target of rapamycin complex 2 and other potential PDK2 kinases phosphorylate AKT at Ser473, resulting in optimal AKT activation [5]. The active phosphorylated AKT then translocates from the cell membrane to other cell compartments to phosphorylate multiple downstream substrates to fulfill its versatile functions. In contrast with canonical AKT activation with coordinate phosphorylation on both Thr308 and Ser473, we identified a novel AKT activation mechanism induced by glucose deprivation by which AKT is selectively phosphorylated on Thr308 but not Ser473, resulting in targeting AKT to a specific group of substrates [3]. In HeLa cells, short-term glucose deprivation (for up to 6 h) induced a modest increase in AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation (16 h) induced a marked increase in AKT phosphorylation at Thr308 (up to 30-fold) but only a modest increase at Ser473 (twofold to threefold). Phosphorylation at Thr308 continued to increase over a 16-h glucose deprivation period, whereas Ser473 phosphorylation peaked at about 6 h and subsequently declined (Figure). Apparently, at least two independent processes are responsible for AKT phosphorylation during 16-h glucose deprivation, with a switch occurring between 6 and 8 h. Our data indicated that the first process takes place primarily via the release of feedback inhibition from p70S6K that results in coordinated phosphorylation of AKT at both Thr308 and Ser473. The second process likely occurs via the formation of a complex including GRP78, PDK1, and AKT that promotes selective AKT phosphorylation at Thr308. Figure AKT phosphorylation on Thr308 and Ser473 induced by glucose deprivation and a proposed in vivo metabolic stress model AKT is functionally activated by selective Thr308 phosphorylation during prolonged glucose deprivation as indicated by increased phosphorylation of several known AKT substrates, including glycogen synthase kinase 3, mammalian target of rapamycin, and Y-box binding protein 1. Strikingly, several well-established AKT substrates, including PRAS40 and BAD, were not targeted by AKT under glucose deprivation conditions. Thus, with selective Thr308 phosphorylation, AKT may have altered substrate selectivity. We also observed coordinate phosphorylation of multiple residues other than Thr308 and Ser473 on single AKT molecules in an isoform-specific manner [6], which may also contribute to substrate selectivity. Therefore, AKT could be shrewdly activated for the right substrate spectrum under different cellular contexts, enabling cells to specifically survive energy crisis during metabolic stress. Shrewd AKT activation may occur in vivo. When a tumor grows, some tumor cells located away from blood vessels may be subjected to glucose deprivation (Figure). We propose that these cells employ a layered defense against cell death according to the severity and duration of metabolic stress. Mild or transient metabolic stress (e.g., transient glucose deprivation) induces modest AKT phosphorylation at both Thr308 and Ser473, which provides the first line of defense against cell death. However, during prolonged glucose deprivation, this line of defense becomes insufficient to protect cells against death and is shut off. The next tier of the survival mechanism is then activated. It selectively and markedly increases AKT phosphorylation at Thr308, providing a second line of defense against cell death caused by severe metabolic stress. Therefore, cells are capable of shifting gears to survive specific harsh conditions. AKT, with its versatile activation mechanisms, provides a set of important survival gears that cells may use under specific conditions. In summary, glucose deprivation induces site- specific phosphorylation and substrate-specific activation of AKT via distinct mechanisms according to the duration of metabolic stress. These mechanisms may explain how cells tolerate metabolic stress in tumors by using AKT as a survival tool. Our findings revealed a novel AKT-mediated survival mechanism under prolonged metabolic stress that is important to the development and implementation of drugs targeting cell metabolism and AKT signaling.

419 Background: Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer, and anti-angiogenic therapy is the current mainstay of treatment. RCC patients develop innate or adaptive resistance to anti-angiogenic therapy. There is a need to identify biomarkers which predict therapeutic resistance and guide combination therapy. Methods: Tissue microarrays with triplicate cores for each case were generated from 33 unaffected kidneys, 41 untreated primary RCC, and 42 bevacizumab and 39 sunitinib pretreated primary RCC from patients with metastatic RCC. Immunohistochemistry was used to visualize immune cell infiltration. Staining quantitation was performed using a Vectra multispectral system. Statistical analysis was performed using unpaired Student´s t-test. Results: We assessed the interaction between anti-angiogenic therapy and tumor immune microenvironment, and determined their impact on clinical outcome. Here we found that anti-angiogenic therapy treated RCC primary tumors demonstrated increased infiltration of CD4+ and CD8+ T lymphocytes, which was inversely related to patient survival. Furthermore, anti-angiogenic therapy treated specimens showed higher infiltration of CD4+FOXP3+regulatory T cells (Tregs) and enhanced expression of checkpoint ligand programed death-ligand 1 (PD-L1). Both immunosuppressive features were correlated with T lymphocyte infiltration and were negatively related to patient overall survival (OS) and/or progression free survival (PFS). Treatment of RCC cell lines and RCC xenografts in immunodeficient mice with sunitinib also increased tumor PD-L1 expression. Conclusions: This study indicates that anti-angiogenic treatment may both positively and negatively regulate the tumor immune microenvironment. These findings generate hypotheses on resistance mechanisms to antiangiogenic therapy, and will guide the development of combination therapy with PD-1/PD-L1 blocking agents.

Hemangioblastoma of the central nervous system is the most common manifestation of Von Hippel-Lindau (VHL) disease, an autosomal dominant inherited cancer syndrome. The underlying molecular mechanism of hemangioblastoma is not known. To discover the molecular drivers of hemangioblastomas, 17 hemangioblastoma human tissue cases were submitted to MIP (molecular inversion probe) analysis to assess copy number alteration. Surprisingly, a focal loss of chromosome 17q was observed in 15 of 17 cases. Further analysis discovered a specific deletion involving HNF1B (Hepatocyte Nuclear Factor 1 homeobox B), a nuclear transcription factor. To elucidate the molecular effects of HNF1B loss, we used three different sequences of shRNA to knock down Hnf1b in mouse embryonic fibroblast (MEF) cells. Loss of Hnf1b resulted in significantly fewer cells arrested in G2/M in response to nocodazole treatment. Cell cycle related protein levels such as cyclin B1, cyclin D1 and phospho-Tyr15-Cdk1 were correspondingly changed. Of note, the cyclin dependent kinase inhibitor Cdkn1b (p27) was remarkably suppressed while another cyclin dependent kinase inhibitor, p21, was not affected at all. To elucidate the mechanism of Hnf1b dependent p27 regulation, the proteasome inhibitor MG132 was used to treat Hnf1b knockdown cells, which failed to increase p27 levels. An in vitro ubiquitination assay demonstrated that Hnf1b did not affect p27 ubiquitination. Both results indicated that Hnf1b regulation of p27 is not via posttranslational ubiquitin-proteasome system. Real time-polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) experiments demonstrated that Hnf1b regulated p27 at the mRNA level and specific consensus binding regions were identified. We then showed that p27 overexpression by lenti-virus infection in Hnf1b knockdown cells normalized the percentage of G2/M arrested cells. Hemangioblastoma are highly vascularized tumors. We discovered a dramatic up-regulation of vascular endothelial growth factor (Vegf) by enzyme-linked immunosorbent assay in cells with Hnf1b knockdown. These findings provided further evidence that loss of Hnf1b is one of the major drivers of the hemangioblastoma disease phenotype. Taken together, our findings for the first time identified HNF1B as a major gene loss in hemangioblastoma patients, and provide molecular evidence that Hnf1b transcriptionally regulates cell cycle protein p27, and drives the proangiogenic phenotype. This novel study sheds insights into molecular mechanisms of hemangioblastoma and provides tools to develop prevention and treatment strategies for the disease. Citation Format: Mianen Sun, Federico Monzon, Lijun Zhou, Xiande Liu, Zhiyong Ding, Xuesong Zhang, Shanshan Bai, Peter German, Sarathi Kalra, In Young ‎ Park, Ruhee Dere, Tia Berry, Xuefei Tong, Cheryl Walker, Nizar Tannir, Surena Matin, Gregory Fuller, Ian McCutcheon, Eric Jonasch. Identification of molecular drivers of human hemangioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2224. doi:10.1158/1538-7445.AM2014-2224

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. Here we reported that the hypoxia inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, was in part constitutively degraded by autophagy. HIF2α interacts with autophagy protein LC3, autophagy receptor p62, and lysosome protein LAMP1. Inhibition of autophagy results in HIF2α accumulation, while induction of autophagy promotes HIF2α degradation. The E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) is required for autophagic degradation of HIF2α. There is a complementary interaction between the proteasome and autophagy in HIF2α degradation. Autophagy inactivation in ATG5 knockout cells redirects HIF2α to proteasomal degradation, while proteasome inhibition induced autophagy and favored the HIF2α-p62 interaction. Importantly, we reported that clear cell renal cell carcinoma (ccRCC) was frequently associated with mono-allelic loss and/or mutation of autophagy related gene ATG7, and low expression level of autophagy genes correlated with ccRCC progression. This study sheds light on the anticancer role for autophagy in ccRCC tumorigenesis, and reveals constitutive autophagic degradation of HIF2α as a novel tumor suppression mechanism. Citation Format: Xian-De Liu, Jun Yao, Durga N. Tripathi, Zhiyong Ding, Yi Xu, Mianen Sun, Jiangwei Zhang, Shanshan Bai, Peter German, Anh Hoang, Lijun Zhou, Xuesong Zhang, Claudio J. Conti, Eleni Efstathiou, Tony N. Eissa, Gordon B. Mills, Cheryl L. Walker, Eric Jonasch. Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 312. doi:10.1158/1538-7445.AM2014-312

Aim: Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer. Recently, anti-angiogenic therapy has been widely used for RCC management. The interaction between immune cell infiltration and anti-angiogenic treatment in RCC, and its association with patient survival, is unknown. We assessed the interaction between anti-angiogenic therapy and immune cell infiltration, and determined their impact on clinical outcome. Methods: Tissue microarrays (TMA) with triplicate cores for each case were generated from formalin-fixed, paraffin embedded tissues from 33 unaffected kidneys, 41 untreated primary RCC, 42 bevacizumab and 39 sunitinib pretreated primary RCC from patients with metastatic RCC. Immunohistochemistry was used to evaluate the infiltration of immune cells. Staining quantitation was performed using a Vectra multispectral system (Perkin Elmer, Waltham, MA). Results: Compared with normal kidney tissues, RCC tissues harbor more infiltrated immune cells, including CD3+ T cells, CD4+ T cells, CD8+ T cells, granzyme B+ T cells and CD68+ macrophages (p 0.05), and macrophage infiltration was not associated with patient survival (p > 0.05). Conclusions: Our study shows that anti-angiogenic therapy increases T cell infiltration in primary RCC tumors of a subset of patients with metastatic disease, and this T cell infiltration is associated with worse clinical outcome. These findings will guide further studies on the mechanistic drivers of T cell infiltration and the impact of these cell populations on therapy response. Disclosure: All authors have declared no conflicts of interest.

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated due to the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. While basal AKT2 phosphorylation was sensitive to inhibition of PI3K, basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C199. Citation Format: Huifang Guo, Meng Gao, Yiling Lu, Jiyong Liang, Philip L. Lorenzi, Shanshan Bai, David H. Hawke, Jie Li, Turgut Dogruluk, Kenneth L. Scott, Eric Jonasch, Gordon B. Mills, Zhiyong Ding. Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C199.

The serine/threonine kinase AKT is a key mediator of cancer cell survival. We demonstrate that transient glucose deprivation modestly induces AKT phosphorylation at both Thr308 and Ser473. In contrast, prolonged glucose deprivation induces selective AKT phosphorylation at Thr308 and phosphorylation of a distinct subset of AKT downstream targets leading to cell survival under metabolic stress. Glucose deprivation-induced AKT Thr308 phosphorylation is dependent on PDK1 and PI3K but not EGFR or IGF1R. Prolonged glucose-deprivation induces the formation of a complex of AKT, PDK1, and the GRP78 chaperone protein, directing phosphorylation of AKT at Thr308 but not Ser473. Our results reveal a novel mechanism of AKT activation under prolonged metabolic stress that protects cells from metabolic stress. The selective activation of AKT phosphorylation at Thr308 that occurs during prolonged nutrient deprivation may provide an unexpected opportunity for the development and implementation of drugs targeting cell metabolism and aberrant AKT signaling. Citation Format: Meng Gao, Jiyong Liang, Yiling Lu, Huifang Guo, Peter German, Shanshan Bai, Eric Jonasch, Xingsheng Yang, Gordon B. Mills, Zhiyong Ding. Site specific activation of AKT protects cells from death induced by glucose deprivation . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-35. doi:10.1158/1538-7445.AM2013-LB-35

369 Background: Mutated von Hippel Lindau (VHL) protein causes disease in sporadic renal cell carcinoma (RCC), and in the eponymous hereditary VHL disease. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL, and potentially reverse the disease phenotype. Methods: The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed. Results: The proteasome inhibitors bortezomib and MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. Thiostrepton has also been recognized as having proteasome inhibitory effects. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds, additionally suggesting unique effects of each of the agents on VHL homeostasis. Conclusions: We have successfully identified compounds that stabilize mutated VHL, including several proteasome inhibitors. It is possible that proteasome inhibition will have a specific clinical effect on patients with point-mutated pVHL. Further work is currently underway to further test this hypothesis in animal xenograft studies using cell lines containing various mutated VHL isoforms, and clinical trials using next-generation proteasome inhibitors are being explored.

Von Hippel-Lindau (VHL) germ line and somatic mutations play determining roles in renal-cell carcinomas (RCC) as well as other diseases, including hemangioblastomas and pheochromocytomas. Although molecules upregulated as a consequence of VHL deficiency have been employed as drug targets in RCC cancer therapy, modulation of pVHL itself has not been explored as a therapeutic strategy in the treatment of VHL derived diseases. Approximately one third of VHL mutations are point mutations, which destabilize pVHL and decrease pVHL intracellular levels, despite in some cases possessing residual functionality. Therefore, interventions that restabilize mutated pVHL may restore physiological protein levels and function. We established a high-throughput screening system to identify small molecules that alter VHL stability. We fused an unstable W117A VHL mutant to a fluorescent protein Venus and transduced the VHL null 786-0 RCC cell line. The fluorescence and protein levels of W117A VHL-Venus were low in untreated cells, and increased significantly after treatment with the proteasome inhibitors MG132 or bortezomib. The fluorescence level served as a VHL protein stability readout for the screen. We then performed a screen on the Prestwick drug library and identified multiple candidates that stabilized VHL protein. Certain candidates have been shown to functionally regulate VHL stability as well as its downstream effecter HIF1a. Our work shows that the high-throughput screening system we established could efficiently identify small molecules that alter VHL stability and functionality, and facilitates exploring VHL as a drug target in the treatment of RCC as well as other VHL diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2011-1379

Finasteride, a specific inhibitor of SRD5A2, one of three isoenzymes of 5α-reductase, reduced prostate cancer incidence by 25% in the Prostate Cancer Prevention Trial, but the molecular mechanisms of its effect remain undelineated. Androgen receptor (AR) signaling is important during prostate cancer pathogenesis and progression, and 5α-reductase converts testosterone to dihydrotestosterone (DHT). We investigated how androgen regulates expression of isoenzymes of 5α-reductase encoded by different genes—SRD5A1, SRD5A2, and SRD5A3—and in turn how finasteride may affect this regulation. By using different prostate cell lines, we found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type-specific manner. This regulation, we demonstrated, requires AR and occurs at the transcriptional level. Moreover, we found that finasteride affects androgen regulation of 5α-reductase expression in a cell type- and AR status-dependent manner. Finasteride counteracts testosterone-driven but not dihydrotestosterone-driven regulation of 5α-reductase expression in LAPC-4 cells, which express wild type AR. However, finasteride does not affect testosterone or dihydrotestosterone regulation of 5α-reductase expression in LNCaP cells, which express a mutant AR (T877A). Our findings suggest that dihydrotestosterone is the major regulator of 5α-reductase expression and that finasteride may produce additional antitumor effects through other mechanisms besides inhibiting 5α-reductase activity. The differential expression levels of 5α-reductase isoenzymes may be one of the factors conferring response or resistance to finasteride treatment and thus may prove significant in prostate cancer prevention. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A105.