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Cancer immunoediting drives the adaptation of tumor cells to host immune surveillance. Immunoediting driven by antigen (Ag)-specific T cells enriches NANOG expression in tumor cells, resulting in a stem-like phenotype and immune resistance. Here, we identify HDAC1 as a key mediator of the NANOG-associated phenotype. NANOG upregulated HDAC1 through promoter occupancy, thereby decreasing histone H3 acetylation on K14 and K27. NANOG-dependent, HDAC1-driven epigenetic silencing of cell-cycle inhibitors CDKN2D and CDKN1B induced stem-like features. Silencing of TRIM17 and NOXA induced immune and drug resistance in tumor cells by increasing antiapoptotic MCL1. Importantly, HDAC inhibition synergized with Ag-specific adoptive T-cell therapy to control immune refractory cancers. Our results reveal that NANOG influences the epigenetic state of tumor cells via HDAC1, and they encourage a rational application of epigenetic modulators and immunotherapy in treatment of NANOG+ refractory cancer types. Cancer Res; 77(18); 5039–53. ©2017 AACR.

Supplementary Figure 2. Immune selection induces HIF-1α in CaSki human cancer cell.

Supplementary Figure 1. SCP3 overexpression induces NANOG-mediated immune resistance and stem-like properties in human cancer cells. Supplementary Figure 2. MHC class I expression and CTL activation capacity of tumor cells are unaffected by SCP3 expression. Supplementary Figure 3. SCP3 expression in tumor cells does not control the viability of CTLs. Supplementary Figure 4. The SCP3 axis is conserved across various human cancer types. Supplementary Figure 5. CDK4/6 inhibitor is more effective than other drugs in SCP3-positive tumor cells. Supplementary Figure 6. Schematic interpretation of therapeutic resistance and tumorigenicity through the SCP3 axis.

Supplementary Figure 1. VEGFA mRNA is up-regulated in human cancer cells subjected to immune selection.

Supplementary Figure 3. Silencing of HIF-1α restores sensitivity of immune-resistant human cancer cells to lysis by tumor-specific human CTLs.

Supplementary Figure 1. Immune-edited CaSki P3 cells showed a stem-like phenotype. Supplementary Figure 2. NANOG does not effect on acetylation of histone H4. Supplementary Figure 3. Expression of HDAC genes, and genes involved in negative regulators of MCL-1 protein by NANOG-HDAC1 axis. Supplementary Figure 4. The NANOG-HDAC1 axis controls immune-resistance, drug-resistance and stem-like property in multiple types of human cancer cells. Supplementary Figure 5. Generation of MDA-MB231 P3 immune-resistant cell lines. Supplementary Figure 6. Regulation of HAT genes by NANOG-HDAC1 axis. Supplementary Figure 7. Schematic interpretation of immune selection-mediated epigenetic change and implications of epigenetic therapy.

Cell lines, Western blot (WB) analysis, Luciferase assay, Trypan blue exclusion assay, Immunohistochemistry, ChIP and quantitative ChIP (qChIP) assays, SUPPLEMENTARY REFERENCES.

Purpose: Adaptation to host immune surveillance is now recognized as a hallmark of cancer onset and progression, and represents an early, indispensable event in cancer evolution. This process of evolution is first instigated by an immune selection pressure imposed by natural host surveillance mechanisms and may then be propagated by vaccination or other types of immunotherapy.Experimental Design: We developed a system to simulate cancer evolution in a live host and to dissect the mechanisms responsible for adaptation to immune selection. Here, we show that the oxygen-sensitive α subunit of hypoxia-inducible factor 1 (HIF-1α) plays a central role in cancer immune adaptation under conditions of normal oxygen tension.Results: We found that tumor cells gain HIF-1α in the course of immune selection under normoxia and that HIF-1α renders tumor cells resistant to lysis by tumor-specific cytotoxic T lymphocytes (CTL) in culture and in mice. The effects of HIF-1α on immune adaptation were mediated through VEGFA-dependent activation of the AKT and ERK signaling pathways, which induced an antiapoptotic gene expression network in tumor cells.Conclusions: Our study therefore establishes a link between immune selection, overexpression of HIF-1α, and cancer immune adaptation under normoxia, providing new opportunities for molecular intervention in patients with cancer. Clin Cancer Res; 21(6); 1438–46. ©2015 AACR.

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Supplementary Figure 3 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

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Supplementary Figure 5 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

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PDF file - 1008KB, mRNA and protein expression of Api5 in TC-1/P0 and TC-1/P3 A17 cells (S1). Api5 expression in murine tumors and their immune resistance (S2). In vitro Generation and Characterization of KKM MART1-specific CTL clone (S3). Characterization of antigen presenting capacity of A375/no insert and A375/API5 cells in vitro (S4). API5 activates FGFR1 signaling pathway (S5).

Supplementary Figure 4 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

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Supplementary Table 1 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

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Supplementary Table 2 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

The skin exhibits tremendous regenerative potential, as different types of progenitor and stem cells regulate skin homeostasis and damage. However, in vitro primary keratinocytes present with several drawbacks, such as high donor variability, short lifespan, and limited donor tissue availability. Therefore, more stable primary keratinocytes are needed to generate multiple uniform in vitro and in vivo skin models.We identified epidermal progenitor cells from primary keratinocytes using Integrin beta 1 (ITGB1) an epidermal stem cell marker markedly decreased after senescence in vitro. Epidermal progenitor cells exhibited unlimited proliferation and the potential for multipotent differentiation capacity. Moreover, they could completely differentiate to form an organotypic skin model including conversed mesenchymal cells in the dermis and could mimic the morphologic and biochemical processes of human epidermis. We also discovered that proliferation and the multipotent differentiation capacity of these cells relied on ITGB1 expression. Eventually, we examined the in vitro and in vivo wound healing capacity of these epidermal progenitor cells.Overall, the findings suggest that these stable and reproducible cells can differentiate into multiple lineages, including human skin models. They are a potentially powerful tool for studying skin regeneration, skin diseases, and are an alternative for in vivo experiments.

Synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, has been implicated in cancer progression, and therapeutic resistance, as well as cancer stem cell (CSC)-like properties. Previously, we demonstrated that SCP3 promotes these aggressive phenotypes via hyperactivation of the AKT signaling pathway, however, the underlying mechanisms responsible for SCP3-induced AKT activation remain to be elucidated. In this study, we demonstrated that the EGF-EGFR axis is the primary route through which SCP3 acts to activate AKT signaling. SCP3 triggers the EGFR-AKT pathway through transcriptional activation of EGF. Notably, neutralization of secreted EGF by its specific monoclonal antibody reversed SCP3-mediated aggressive phenotypes with a concomitant reversal of EGFR-AKT activation. In an effort to elucidate the molecular mechanisms underlying SCP3-induced transcriptional activation of EGF, we identified Jun activation domain-binding protein 1 (JAB1) as a binding partner of SCP3 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that SCP3 induces EGF transcription through physical interaction with JAB1. Thus, our findings establish a firm molecular link among SCP3, EGFR, and AKT by identifying the novel roles of SCP3 in transcriptional regulation. We believe that these findings hold important implications for controlling SCP3high therapeutic-refractory cancer.

BACKGROUND: The medial hamstring (MH) and lateral hamstring (LH) can be selectively trained through tibial internal and external rotation during prone knee flexion. However, no study has identified how a combined tibial rotation and lumbo-pelvic stability strategy influences MH and LH muscle activities. OBJECTIVE: To investigate the combined effects of tibial rotation and the abdominal drawing-in maneuver (ADIM) on MH and LH muscle activities as well as pelvic rotation during prone knee flexion. METHODS: Fifteen female volunteers performed prone knee flexion with tibial internal and external rotation, with and without the ADIM. Under each condition, MH and LH muscle activities were measured by surface electromyography (EMG), and the pelvic rotation angle by a smartphone inclinometer application. RESULTS: The results showed increased MH (without the ADIM: p< 0.001, effect size (d) = 2.05; with the ADIM: p< 0.001, d= 1.71) and LH (without the ADIM: p< 0.001, d= 1.64; with the ADIM: p= 0.001, d= 1.58) muscle activities under internal and external tibial rotation, respectively. However, addition of the ADIM led to increased MH (internal tibial rotation: p= 0.001, d= 0.67; external tibial rotation: p= 0.019, d= 0.45) and LH (internal tibial rotation: p= 0.003, d= 0.79; external tibial rotation: p< 0.001, d= 1.05) muscle activities combined with reduced pelvic rotation (internal tibial rotation: p< 0.001, d= 3.45; external tibial rotation: p< 0.001, d= 3.01) during prone knee flexion. CONCLUSIONS: These findings suggest that the ADIM could be useful for reducing compensatory pelvic rotation and enhancing selective muscle activation in the MH and LH, according to the direction of tibial rotation, during prone knee flexion.

Progranulin (PGRN) is a cysteine-rich secreted protein expressed in endothelial cells, immune cells, neurons, and adipocytes. It was first identified for its growth factor-like properties, being implicated in tissue remodeling, development, inflammation, and protein homeostasis. However, these findings are controversial, and the role of PGRN in liver disease remains unknown. In the current study, we examined the effect of PGRN in two different models of chronic liver disease, methionine‐choline‐deficient diet (MCD)-induced non-alcoholic steatohepatitis (NASH) and carbon tetrachloride (CCl4)-induced liver fibrosis. To induce long-term expression of PGRN, PGRN-expressing adenovirus was delivered via injection into the tibialis anterior. In the CCl4-induced fibrosis model, PGRN showed protective effects against hepatic injury, inflammation, and fibrosis via inhibition of nuclear transcription factor kappa B (NF-κB) phosphorylation. PGRN also decreased lipid accumulation and inhibited pro-inflammatory cytokine production and fibrosis in the MCD-induced NASH model. In vitro treatment of primary macrophages and Raw 264.7 cells with conditioned media from hepatocytes pre-treated with PGRN prior to stimulation with tumor necrosis factor (TNF)-α or palmitate decreased their expression of pro-inflammatory genes. Furthermore, PGRN suppressed inflammatory and fibrotic gene expression in a cell culture model of hepatocyte injury and primary stellate cell activation. These observations increase our understanding of the role of PGRN in liver injury and suggest PGRN delivery as a potential therapeutic strategy in chronic inflammatory liver disease.

Hepatitis B virus (HBV) X protein (HBx) is associated with hepatocarcinogenesis. E2-EPF ubiquitin carrier protein (UCP) catalyzes ubiquitination of itself and von Hippel–Lindau protein (pVHL) for degradation and associates with tumor growth and metastasis. However, it remains unknown whether HBx modulates the enzyme activity of UCP and thereby influences hepatocarcinogenesis. Here, we show that UCP is highly expressed in liver tissues of HBx-transgenic mice, but not non-transgenic mice. UCP was more frequently expressed in HBV-positive liver cancers than in HBV-negative liver cancers. HBx binds to UCP specifically and serotype independently, and forms a ternary complex with UCP and pVHL. HBx inhibits self-ubiquitination of UCP, but enhances UCP-mediated pVHL ubiquitination, resulting in stabilization of hypoxia-inducible factor-1α and -2α. HBx and UCP stabilize each other by mutually inhibiting their ubiquitination. HBx promotes cellular proliferation and metastasis via UCP. Our findings suggest that UCP plays a key role in HBV-related hepatocarcinogenesis. Electronic supplementary material The online version of this article (10.1007/s00018-019-03066-9) contains supplementary material, which is available to authorized users.

Accurate prediction of pharmacokinetic (PK) and pharmacodynamic (PD) characteristics is critical for drug development. Oral drugs are particularly difficult because they are absorbed by the intestine and metabolized in the liver before systemic metabolism in vivo; this is called the first-pass effect and is a critical factor for predicting oral bioavailability (BA). Here, we fabricated a new networking and circulating cell culture system (NCCS), mimicking the circulatory system and interaction of organs for studying the pharmacokinetic and pharmacodynamics of oral drugs in vitro. NCCS consisted of a micro-pump for circulating fluids, two types of multi-insert culture dishes for culturing different cell types, and an orbital shaker for mixing; flow rate and shaking-speed were controlled by weight-sensors and drivers. A first-pass effect test was performed using functionally differentiated HepaRG and Caco-2 cell lines, using a new modified spheroid forming unit (SFU) protocol. To verify the similarity of PK (first-pass effect) data of NCCS with the data from the human body, 15 reference drugs were chosen and their associated data were obtained by liquid chromatography-mass spectrometry analysis. NCCS generated absorption and metabolism data showed >70% similarity to human data respectively. NCCS can also be used to demonstrate species differences. Animal models are the primary basis for drug discovery, development, and testing. However, the weak correlation between humans and animals, particularly regarding absorption and metabolism, is a substantial limitation for the use of animal models. Here we compare human and mouse acetaminophen (APAP) metabolism using NCCS, and its application can be extended to assess cellular responses, such as efficacy and toxicity, simultaneously.

Cancer cachexia is a highly debilitating condition characterized by weight loss and muscle wasting that contributes significantly to the morbidity and mortality of pancreatic cancer. The factors that induce cachexia in pancreatic cancer are largely unknown. We previously showed that pancreatic adenocarcinoma upregulated factor (PAUF) secreted by pancreatic cancer cells is responsible for tumor growth and metastasis. Here, we analyzed the relation between pancreatic cancer-derived PAUF and cancer cachexia in mice and its clinical significance. Body weight loss and muscle weight loss were significantly higher in mice with Panc-1/PAUF tumors than in those with Panc-1/Mock tumors. Direct administration of rPAUF to muscle recapitulated tumor-induced atrophy, and a PAUF-neutralizing antibody abrogated tumor-induced muscle wasting in Panc-1/PAUF tumor-bearing mice. C2C12 myotubes treated with rPAUF exhibited rapid inactivation of Akt-Foxo3a signaling, resulting in Atrogin1/MAFbx upregulation, myosin heavy chain loss, and muscle atrophy. The neutrophil-to-lymphocyte ratio and body weight loss were significantly higher in pancreatic cancer patients with high PAUF expression than in those with low PAUF expression. Analysis of different pancreatic cancer datasets showed that PAUF expression was significantly higher in the pancreatic cancer group than in the nontumor group. Analysis of The Cancer Genome Atlas data found associations between high PAUF expression or a high DNA copy number and poor overall survival. Our data identified tumor-secreted circulating PAUF as a key factor of cachexia, causing muscle wasting in mice. Neutralizing PAUF may be a useful therapeutic strategy for the treatment of pancreatic cancer-induced cachexia., Pancreatic cancer: Protein promotes cancer-related weight loss A protein involved in tumor growth is also a likely cause of weight loss during pancreatic cancer, and may be a useful target for prognosis and treatment. Pancreatic adenocarcinoma upregulated factor (PAUF) is secreted by pancreatic cancer cells and is also found in other cancers where significant weight loss occurs. Wonbeak Yoo, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea, and co-workers examined the role of PAUF in cancer-related weight loss in mice. Mice inoculated with PAUF-expressing cancer cells experienced weight loss despite no change in food consumption. PAUF increased muscle atrophy and triggered muscle wasting pathways. PAUF is routinely measured in cancer patients, and could therefore provide a convenient biomarker to inform treatment decisions. The researchers suggest that neutralising PAUF could be a valuable therapeutic option.

Background Intracellular lipid accumulation is associated with various diseases, particularly cancer. Mitochondrial dysfunction is considered as a cause of lipid accumulation; however, the related underlying mechanism remains unclear. Findings We found that Von Hippel-Lindau (VHL)-deficiency led to lipid accumulation and mitochondrial dysfunction in renal cell carcinoma cells. Moreover, VHL downregulated ATP-citrate lyase (ACLY), a key enzyme in de novo lipid synthesis, at the transcriptional level, which inhibited intracellular lipid accumulation in human renal carcinoma tissues. We identified PPARγ as the transcription factor regulating ACLY expression by binding to the cis-regulatory site PPRE on its promoter. VHL directly interacted with and promoted ubiquitination of PPARγ, leading to its degradation both in vitro and in vivo, resulting in the downregulation of ACLY. Furthermore, adenovirus-mediated VHL overexpression substantially ameliorated hepatic steatosis induced by a high-fat diet in db/db mice. Importantly, low VHL expression was associated with high ACLY expression and poor prognosis in human liver carcinoma in a dataset in The Cancer Genome Atlas. Conclusions VHL plays role in cellular lipid metabolism via regulating mitochondria and targeting PPARγ, a transcription factor for ACLY independent of hypoxia-inducible factor 1α. A novel VHL-PPARγ-ACLY axis and its implication in fatty liver disease and cancer were uncovered.

The spastin protein (SPAST) contains an ATPase with diverse cellular activities (AAA) domain and regulates microtubule dynamics. Missense mutations of the SPAST gene are frequently detected in patients with hereditary spastic paraplegias (HSPs) and represent the main reason of loss of SPAST function; however, the pathogenicity of mutant SPAST is heterogeneous. Here, SPAST variant with an I344K mutation (I344K-SPAST) was identified in a Korean family with autosomal dominant-type HSP. We investigated the role of the I344K-SPAST in HSP to provide a therapeutic mechanism. The I344K-SPAST mutation prolonged the half-life of the protein compared to wild-type SPAST (WT-SPAST) in cells by modulating post-translational modifications for proteasomal degradation. I344K-SPAST was localized in microtubule but defective in microtubule severing and ATPase activity compared to WT-SPAST in vitro and in cells. Mutant M87 isoform harboring the same mutation with I344K-M1 SPAST also increased protein stability and loss of MT severing activity, but the pathogenicity was not stronger than I344K-M1 SPAST in neurite outgrowth. Overexpression of I344K-SPAST resulted in microtubule accumulation following inhibited neurite growth in neuroblastoma, neural progenitor cells and mouse primary cortical neurons. Conversely, these pathogenic effects of I344K-SPAST were reduced by overexpression of WT-M1 SPAST in a dose dependent manner since WT-SPAST could interact with I344K-SPAST. Our data therefore provide proof-of-concept that gene transfer of WT-M1 SPAST may serve as a valid therapeutic option for HSPs.

Anti-vascular endothelial growth factor (VEGF) therapy has demonstrated efficacy in treating human metastatic cancers, but therapeutic resistance is a practical limitation and most tumors eventually become unresponsive. To identify microenvironmental factors underlying the resistance of cancer to antiangiogenesis therapy, we conducted genomic analyses of intraperitoneal ovarian tumors in which adaptive resistance to anti-VEGF therapy (B20 antibody) developed. We found that expression of the microseminoprotein, prostate-associated (MSMP) gene was substantially upregulated in resistant compared with control tumors. MSMP secretion from cancer cells was induced by hypoxia, triggering MAPK signaling in endothelial cells to promote tube formation in vitro. Recruitment of the transcriptional repressor CCCTC-binding factor (CTCF) to the MSMP enhancer region was decreased by histone acetylation under hypoxic conditions in cancer cells. MSMP siRNA, delivered in vivo using the DOPC nanoliposomes, restored tumor sensitivity to anti-VEGF therapy. In ovarian cancer patients treated with bevacizumab, serum MSMP concentration increased significantly only in non-responders. These findings imply that MSMP inhibition combined with the use of antiangiogenesis drugs may be a new strategy to overcome resistance to antiangiogenesis therapy.

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial–mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

Background The interaction of vascular endothelial growth factor-C (VEGF-C)/VEGF-D/VEGF receptor-3 is considered to be a major driver of lymphangiogenesis, however the mechanism of this process remains unclear. We aimed to investigate the possible lymphangiogenic significance of synaptonemal complex protein 3 (SCP3) in non-small cell lung cancer (NSCLC). Methods The expression of SCP3, VEGF-C, and VEGF-D were measured and examined a correlation between SCP3 and VEGF-C or VEGF-D in various human lung cancer cell lines. Subsequently, we assessed SCP3, VEGF-A, VEGF-B, VEGF-C, and VEGF-D expression in archival tumor tissues from 89 NSCLC patients with lymph node (LN) metastasis by combined immunohistochemistry with quantitative digital image analysis. Results Positive correlations between SCP3 and VEGF-C expression (R 2 = 0.743) and VEGF-D expression (R 2 = 0.932) were detected in various human lung cancer cell lines. The high expression of SCP3, VEGF-A, VEGF-B, VEGF-C, and VEGF-D were detected in 24 (27.0%), 22 (24.7%), 27 (30.3%), 27 (30.3%), and 24 cases (27.0%), respectively. Notably, SCP3 positively correlated with VEGF-C and VEGF-D expression (for both, P

Stable and reproducible kidney cellular models could accelerate our understanding of diseases, help therapeutics development, and improve nephrotoxicity screenings. Generation of a reproducible in vitro kidney models has been challenging owing to the cellular heterogeneity and structural complexity of the kidney. We generated mixed immortalized cell lines that stably maintained their characteristic expression of renal epithelial progenitor markers for the different lineages of kidney cellular compartments via the BMP7 signaling pathway from a mouse and a human whole kidney. These cells were used to generate functional and matured kidney spheroids containing multiple renal lineages, such as the proximal tubule, loop of Henle, distal tubules, and podocytes, using extracellular matrix and physiological force, named spheroid-forming unit (SFU). They expressed all apical and basolateral transporters that are important for drug metabolism and displayed key functional aspects of the proximal tubule, including protein endocytosis and increased gamma-glutamyltransferase activity, and cyclic AMP responded to external cues, such as parathyroid hormone. Following exposure, cells fluxed and took up drugs via proximal tubule-specific apical or basolateral transporters, and displayed increased cell death and expression of renal injury marker. Here, we developed a new differentiation method to generate kidney spheroids that structurally recapitulate important features of the kidney effectively and reproducibly using mixed immortalized renal cells, and showed their application for renal toxicity studies.

Yes-associated protein (YAP)-1 is highly upregulated in pancreatic cancer and associated with tumor progression. However, little is known about the role of YAP1 and related genes in pancreatic cancer. Here, we identified target genes regulated by YAP1 and explored their role in pancreatic cancer progression and the related clinical implications. Analysis of different pancreatic cancer databases showed that Neuromedin U (NMU) expression was positively correlated with YAP1 expression in the tumor group. The Cancer Genome Atlas data indicated that high YAP1 and NMU expression levels were associated with poor mean and overall survival. YAP1 overexpression induced NMU expression and transcription and promoted cell motility in vitro and tumor metastasis in vivo via upregulation of epithelial&ndash, mesenchymal transition (EMT), whereas specific inhibition of NMU in cells stably expressing YAP1 had the opposite effect in vitro and in vivo. To define this functional association, we identified a transcriptional enhanced associate domain (TEAD) binding site in the NMU promoter and demonstrated that YAP1&ndash, TEAD binding upstream of the NMU gene regulated its transcription. These results indicate that the identified positive correlation between YAP1 and NMU is a potential novel drug target and biomarker in metastatic pancreatic cancer.

Von Hippel Lindau (VHL) expression is significantly decreased in high-grade RCC, and autophagy, which is involved in tumor growth, invasion, differentiation, and metastasis, is activated in various human cancers. However, the relationship of autophagy and VHL in tumor progression remains controversial. Here, we showed that the expression levels of VHL and microtubule-associated protein 1 light chain 3B (MAP1LC3B, LC3B) were inversely correlated with various tumor grades of RCC tissues. pVHL was found to possess the LIR motif within a beta domain that interacted with MAP1LC3B and ubiquitinated it. The L101A VHL mutant failed to interact with MAP1LC3B, thereby failing to induce ubiquitination. MAP1LC3B-mediated autophagy was inhibited by functional pVHL and the ubiquitination of MAPLC3B was implicated in autophagy-induced cell death. We screened various autophagy inducers to determine the physiological function of the inhibition of LC3B-mediated autophagy by pVHL using VHL-deficient and VHL-expressing cell lines. MLN9708, a proteasome inhibitor, potently induced autophagy via the induction of MAP1LC3B and sensitized the cell to autophagy-mediated cell death in VHL-deficient and VHL-mutant (L101A) cells. In conclusion, our results showed that pVHL interacts with MAPL1LC3B and inhibits LC3B-mediated autophagy via MAP1LC3B ubiquitination. Furthermore, the activation of autophagy by the proteasome inhibitor MLN9708 induced cell death, indicating that MLN9708 can be used for VHL-deficient RCC therapy.

Objective: EGFL6, epidermal growth factor (EGF)-like domain multiple 6, has been found to be an important target for antiangiogenesis approaches. However, its receptor is not fully known and is the focus of the current work. Method: We first isolated endothelial cells from 10 high-grade serous ovarian cancers, 5 normal ovarian tissues, and 7 healing wound patient samples, and searched for genes selectively expressed in tumor endothelial cells. For receptor studies, we performed an unbiased antibody array for human phospho-RTKs with EGFL6 treatment. Results: EGFL6 was found to be highly expressed in tumor endothelial cells, but not in wound endothelial cells. EGFL6 promoted tumor angiogenesis by increasing tube formation and migration ability of tumor endothelial cells via Tie2, which is expressed on tumor endothelial cells. Moreover, we developed new EGFL6-blocking antibodies, which reduced tumor angiogenesis and in vivo tumor growth without impairing wound healing. Human phospho-RTK array showed that EGFR could be a candidate receptor for EGFL6. Furthermore, EGL6 activated EGFR expressed on tumor cells and promoted invasion ability of tumor cells by 2-fold. Interestingly, EGFL6 secreted from tumor endothelial cells increased the mRNA expression of EGFL6 in tumor cells, which suggests that there may be a positive regulatory loop between endothelial and tumor cells. Conclusion: EGFL6 could be a promising target for development of antiangiogenesis therapies. Citation Format: Mana Taki, Kyung Hee Noh, Lingegowda S. Mangala, Ningyan Zhang, Zhiqiang An, Anil K. Sood. The search for EGFL6 receptor: Implications for tumor angiogenesis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B73.

Stable and reproducible kidney cellular models could accelerate disease understanding, help therapeutics development and improve nephrotoxicity screens. Generation of a reproducible in vitro kidney models have been challenging due to the cellular heterogeneity and structural complexity. Here, we established a renal cell line and developed a system to generate kidney spheroids containing multiple cell types that structurally and functionally recapitulates features of the kidney. First we generated immortalized cell lines from mouse and human kidneys that stably maintain their characteristics of expression of renal progenitor markers and its proliferative capacity. We show that these cells can be differentiated into kidney spheroids that contains multiple nephron segments; such as proximal tubule Loop of Henle, distal tubules, and podocytes. The cellular differentiation is enhanced by culturing these structures in an extracellular matrix. They expressed all apical and basolateral transporters that are important for drug metabolism. The spheroids recapitulated key functional aspects of the kidney including protein endocytosis, increased gamma-glutamyltransferase activity and cyclic AMP responded to external cues, such as parathyroid hormone. Following exposure, cells efflux and take up drugs via proximal tubule specific apical or basolateral transporters, display increased cell death and expression of renal injury marker. Here, we generated new mouse and human renal cell lines, developed an in vitro differentiation method to generate kidney spheroids that recapitulate the kidney both structurally and functionally, and can be used for drug toxicity and therapeutics development. Funding Statement: This research was supported by the KRIBB Research Initiative Program and the Midcareer Researcher Program (2016R1A2B4011124) through the National Research Foundation of Korea funded by the Ministry of Science and ICT. Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All experimental protocol was approved by the Institutional Ethics Committee/IRB of the Korea Research Institute of Bioscience and Biotechnology (KRIBB).

The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK+ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK+ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK+ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK+ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK+ NSCLC.

Metastatic recurrence after cancer treatments with radiation, cancer drugs, or even immunotherapeutic agents (cytokine, antibody, lymphocyte etc.) is often intractable and fatal for cancer patients. Therefore, molecular understanding of metastatic recurrence is necessary. Recently, these recurrent and metastatic tumor cells with resistance to cancer drugs have been reported to possess stem-like attributes and epithelial-mesenchymal transition (EMT) phenotype. Previously, we also found that antigen-specific cytotoxic T lymphocyte (CTL)-mediated immunotherapy conferred tumor cells with immune-resistant and stem-like phenotypes by hyper-activating NANOG/TCL1/AKT signaling axis. In this study, we report that these immunoedited cells have high metastatic capability and phenotypes. These cells exhibit enhanced migration, infiltration, and invasiveness in vitro as well as formation of metastatic lung nodules in vivo. Moreover, they display EMT-like features characterized by increased expression of BMI1 and TWIST1. Importantly, these pleiotropic phenotypes of metastasis through the expression of the EMT-associated molecules were critically dependent on the NANOG/TCL1A/AKT signaling axis, which was also conserved across multiple types of human cancer. Thus, we provide proof of the principle that inhibition of the NANOG axis is an effective strategy to control metastasis of immunoedited cancer, particularly, after CTL-based immunotherapy.