Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer

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Abstract

BACKGROUND. Liquid biopsies have demonstrated that the constitutively active androgen receptor splice variant-7 (AR-V7) associates with reduced response and overall survival (OS) from endocrine therapies in castration resistant prostate cancer (CRPC). However, these studies provide little information pertaining to AR-V7 expression in prostate cancer (PC) tissue. METHODS. Following generation and validation of a novel AR-V7 antibody for immunohistochemistry, AR-V7 protein expression was determined for 358 primary prostate samples and 293 metastatic biopsies. Associations with disease progression, full length AR (AR-FL) expression, response to therapy, and gene expression was determined. RESULTS. We demonstrated that AR-V7 protein is rarely expressed (<1%) in primary PC but is frequently detected (75% of cases) following androgen deprivation therapy, with further significant (P = 0.020) increase in expression following abiraterone acetate or enzalutamide therapy. In CRPC, AR-V7 expression is predominantly (94% of cases) nuclear and correlates with AR-FL expression (P ≤ 0.001) and AR copy number (P = 0.026). However, dissociation of expression was observed suggesting mRNA splicing remains crucial for AR-V7 generation. AR-V7 expression was heterogeneous between different metastases from a patient although AR-V7 expression was similar within a metastasis. Moreover, AR-V7 expression correlated with a unique 59-gene signature in CRPC, including HOXB13, a critical co-regulator of AR-V7 function. Finally, AR-V7 negative disease associated with better PSA responses (100% vs 54%; P = 0.03) and OS (74.3 vs 25.2mo, HR 0.23 [0.07-0.79], P = 0.02) from endocrine therapies (pre-chemotherapy). CONCLUSION. This study provides impetus to develop therapies that abrogate AR-V7 signaling to improve our understanding of AR-V7 biology, and to confirm its clinical significance.

Authors

Adam Sharp, Ilsa Coleman, Wei Yuan, Cynthia Sprenger, David Dolling, Daniel Nava Rodrigues, Joshua W. Russo, Ines Figueiredo, Claudia Bertan, George Seed, Ruth Riisnaes, Takuma Uo, Antje Neeb, Jonathan Welti, Colm Morrissey, Suzanne Carreira, Jun Luo, Peter S. Nelson, Steven P. Balk, Lawrence D. True, Johann De Bono, Stephen R. Plymate

microRNA-668 represses MTP18 to preserve mitochondrial dynamics in ischemic acute kidney injury

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Abstract

The pathogenesis of ischemic diseases remains unclear. Here we demonstrate the induction of microRNA-668 (mir-668) in ischemic acute kidney injury (AKI) in human patients, mice, and renal tubular cells. The induction was HIF-1dependant as HIF-1-deficiency in cells and kidney proximal tubules attenuated mir-668 expression. We further identified a functional HIF-1 binding site in mir-668 gene promoter. Anti-mir-668 increased apoptosis in renal tubular cells and enhanced ischemic AKI in mice, whereas mir-668 mimic was protective. Mechanistically, anti-mir-668 induced mitochondrial fragmentation, whereas mir-668 blocked mitochondrial fragmentation during hypoxia. We analyzed mir-668 target genes through immunoprecipitation of microRNA-induced silencing complexes followed by RNA deep sequencing and identified 124 protein-coding genes as likely targets of mir-668. Among these genes, only Mitochondrial Protein 18 KDa (MTP18) has been implicated in mitochondrial dynamics. In renal cells and mouse kidneys, mir-668 mimic suppressed MTP18, whereas anti-mir-668 increased MTP18 expression. Luciferase microRNA target reporter assay further verified MTP18 as a direct target of mir-668. In renal tubular cells, knockdown of MTP18 suppressed mitochondrial fragmentation and apoptosis. Together, the results suggest that mir-668 is induced via HIF-1 in ischemic AKI and, upon induction, mir-668 represses MTP18 to preserve mitochondrial dynamics for renal tubular cell survival and kidney protection.

Authors

Qingqing Wei, Haipeng Sun, Shuwei Song, Yong Liu, Pengyuan Liu, Man J. Livingston, Jianwen Wang, Mingyu Liang, Qing-Sheng Mi, Yuqing Huo, N. Stanley Nahman, Changlin Mei, Zheng Dong

CDCA7 and HELLS mutations undermine nonhomologous end joining in centromeric instability syndrome

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Abstract

Mutations in CDCA7 and HELLS that respectively encode a CXXC-type zinc finger protein and a SNF2 family chromatin remodeler cause immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome type 3 and 4, respectively. Here, we demonstrate that classical non-homologous end joining (C-NHEJ) proteins Ku80 and Ku70, as well as HELLS coimmunoprecipitated with CDCA7. The coimmunoprecipitation of the repair proteins was sensitive to nuclease treatment and an ICF3 mutation in CDCA7 that impairs its chromatin binding. The functional importance of these interactions was strongly suggested by the compromised C-NHEJ activity and significant delay in Ku80 accumulation at DNA damage sites in CDCA7 and HELLS deficient HEK293 cells. Consistent with the repair defect, these cells displayed increased apoptosis, abnormal chromosome segregation, aneuploidy, centrosome amplification, and significant accumulation of γH2AX signals. Although less prominent, cells mutated for the other ICF genes DNMT3B and ZBTB24 (responsible for ICF type 1 and 2, respectively) showed similar defects. Importantly, lymphoblastoid cells from ICF patients shared the same changes detected in the mutant HEK293 cells to varying degrees. Although the C-NHEJ defect alone did not cause CG hypomethylation, CDCA7 and HELLS are involved in maintaining CG methylation at centromeric and pericentromeric repeats. The defect in C-NHEJ may account for some common features of ICF cells, including centromeric instability, abnormal chromosome segregation, and apoptosis.

Authors

Motoko Unoki, Hironori Funabiki, Guillaume Velasco, Claire Francastel, Hiroyuki Sasaki

Osteopontin mediates glioblastoma-associated macrophage infiltration and is a therapeutic target

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Abstract

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration, thus we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immune blotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA and CRISPR/CAS9 techniques followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal GBM show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αVβ5 (ITGαVβ5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived OPN and host-derived OPN was critical for glioma development. OPN deficiency in either the innate immune or glioma cells demonstrated a marked reduction of M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. OPN can be exploited as an immune modulatory target, with efficacious therapeutic results using systemically administered OPN-4-1BB bispecific aptamers, increasing median survival time by 68% (P < 0.05). OPN is an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and can be exploited as a therapeutic target.

Authors

Jun Wei, Anantha Marisetty, Brett Schrand, Konrad Gabrusiewicz, Yuuri Hashimoto, Martina Ott, Zacharia Grami, Ling-Yuan Kong, Xiaoyang Ling, Hillary G. Caruso, Shouhao Zhou, Y. Alan Wang, Gregory N. Fuller, Jason T. Huse, Eli Gilboa, Nannan Kang, Xingxu Huang, Roel Verhaak, Shulin Li, Amy B. Heimberger

Bone marrow transplantation generates T cell–dependent control of myeloma in mice

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Abstract

Transplantation with autologous hematopoietic progenitors remains an important consolidation treatment for multiple myeloma (MM) patients and is thought to prolong disease plateau-phase by providing intensive cytoreduction. However, transplantation induces inflammation in the context of profound lymphodepletion that may cause hitherto unexpected immunological effects. We developed preclinical models of bone marrow transplantation (BMT) for MM using Vk*MYC myeloma-bearing recipients and donors that were myeloma-naïve or were myeloma-experienced to simulate autologous transplantation. Surprisingly, we demonstrate broad induction of T cell-dependent myeloma control, most efficiently from memory T cells within myeloma-experienced grafts, but also through priming of naïve T cells after BMT. CD8+ T cells from mice with controlled myeloma had a distinct TCR repertoire and higher clonotype overlap relative to myeloma-free BMT recipients. Furthermore, T cell-dependent myeloma control could be adoptively transferred to secondary recipients, and was myeloma clone-specific. Interestingly, donor-derived IL-17A acted directly on myeloma cells expressing the IL-17-receptor to induce a transcriptional landscape that promoted tumor growth and immune escape. Conversely, donor IFNγ secretion and signaling was critical to protective immunity, and was profoundly augmented by CD137 agonists. These data provide new insights into the mechanisms of action of transplantation in myeloma and suggests rational approaches to improving clinical outcome.

Authors

Slavica Vuckovic, Simone A. Minnie, David Smith, Kate H. Gartlan, Thomas S. Watkins, Kate A. Markey, Pamela Mukhopadhyay, Camille Guillerey, Rachel D. Kuns, Kelly R. Locke, Antonia L. Pritchard, Peter A. Johansson, Antiopi Varelias, Ping Zhang, Nicholas D. Huntington, Nicola Waddell, Marta Chesi, John J. Miles, Mark J. Smyth, Geoffrey R. Hill

Temporal dynamics of Wnt-dependent transcriptome reveals an oncogenic Wnt/MYC/ribosome axis

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Abstract

Activating mutations in the Wnt pathway drive a variety of cancers, but the specific targets and pathways activated by Wnt ligands are not fully understood. To bridge this knowledge gap, we performed a comprehensive time-course analysis of Wnt-dependent signaling pathways in an orthotopic model of Wnt-addicted pancreatic cancer, using a PORCN inhibitor currently in clinical trials, and validated key results in additional Wnt-addicted models. The temporal analysis of the drug-perturbed transcriptome demonstrated direct and indirect regulation of greater than 3,500 Wnt activated genes (23% of the transcriptome). Regulation was both via Wnt/β-catenin, and through the modulation of protein abundance of important transcription factors including MYC via Wnt/STOP. Our study identifies a central role of Wnt /β-catenin and Wnt/STOP signaling in controlling ribosomal biogenesis, a key driver of cancer proliferation.

Authors

Babita Madan, Nathan Harmston, Gahyathiri Nallan, Alex Montoya, Peter Faull, Enrico Petretto, David M. Virshup

Gα₁₂ ablation exacerbates liver steatosis and obesity by suppressing USP22/SIRT1-regulated mitochondrial respiration

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Abstract

Non-alcoholic fatty liver disease (NAFLD) arises from mitochondrial dysfunction under sustained imbalance between energy intake and expenditure, but the underlying mechanisms controlling mitochondrial respiration have not been entirely understood. Heterotrimeric G proteins converge signals from activated GPCRs, and modulate cell signaling pathways to maintain metabolic homeostasis. Here, we investigated the regulatory role of Gα12 on hepatic lipid metabolism and whole-body energy expenditure in mice. Fasting increased Gα12 level in mouse liver. Gα12 ablation markedly augmented fasting-induced hepatic fat accumulation. cDNA microarray analysis from Gna12 KO liver revealed that Gα12 signaling pathway regulated sirtuin 1 (SIRT1) and PPARα responsible for mitochondrial respiration. Defective induction of SIRT1 upon fasting was observed in the liver of Gna12 KO mice, which was reversed by lentivirus-mediated Gα12 overexpression in hepatocytes. Mechanistically, Gα12 stabilized SIRT1 protein through transcriptional induction of USP22 via HIF-1α increase. Gα12 levels were markedly diminished in liver biopsies from NAFLD patients. Consistently, Gna12 KO mice fed high-fat diet displayed greater susceptibility to diet-induced liver steatosis and obesity due to decrease in energy expenditure. Our results demonstrate that Gα12 regulates SIRT1-dependent mitochondrial respiration through HIF-1α-dependent USP22 induction, identifying Gα12 as an upstream molecule that contributes to the regulation of mitochondrial energy expenditure.

Authors

Tae Hyun Kim, Yoon Mee Yang, Chang Yeob Han, Ja Hyun Koo, Hyunhee Oh, Su Sung Kim, Byoung Hoon You, Young Hee Choi, Tae-Sik Park, Chang Ho Lee, Hitoshi Kurose, Mazen Noureddin, Ekihiro Seki, Yu-Jui Yvonne Wan, Cheol Soo Choi, Sang Geon Kim

Notch signaling suppresses glucose metabolism in mesenchymal progenitors to restrict osteoblast differentiation

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Abstract

Notch signaling critically controls cell fate decisions in mammals, both during embryogenesis and in adults. In the skeleton, Notch suppresses osteoblast differentiation and sustains bone marrow mesenchymal progenitors during postnatal life. Stabilizing mutations of Notch2 cause the Hajdu-Cheney syndrome characterized by early onset osteoporosis in humans, but the mechanism whereby Notch inhibits bone accretion is not fully understood. Here we report that activation of Notch signaling by either Jagged1 or Notch2 intracellular domain suppresses glucose metabolism and osteoblast differentiation in primary cultures of bone marrow mesenchymal progenitors. Importantly, deletion of Notch2 in the limb mesenchyme increases both glycolysis and bone formation in the long bones of postnatal mice, whereas pharmacological reduction of glycolysis abrogates the excessive bone formation. Mechanistically, Notch reduces the expression of glycolytic and mitochondrial Complex I genes, resulting in a decease in mitochondrial respiration, superoxide production and Ampk activity. Forced activation of Ampk restores glycolysis in the face of Notch signaling. Thus, suppression of glucose metabolism contributes to the mechanism whereby Notch restricts osteoblastogenesis from bone marrow mesenchymal progenitors.

Authors

Seung-Yon Lee, Fanxin Long

Cigarette smoke and HIV synergistically affect lung pathology in cynomolgus macaques

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Abstract

In the era of combined antiretroviral therapy (cART), lung diseases such as chronic bronchitis (CB) and COPD are common among persons living with HIV (PLWH), particularly smokers. Although smoking is highly prevalent among PLWH, HIV may be an independent risk factor for lung diseases; however, the role of HIV and cigarette smoke (CS) and their potential interaction in the development of chronic lung diseases among PLWH has not been delineated. To investigate this interaction, cynomolgus macaques were exposed to CS and/or simian-adapted human immunodeficiency virus (SHIV) and treated with cART. The development of CB and the lung functions were evaluated following CS±SHIV treatment. The results showed that in the lung, SHIV was a strong independent risk factor for goblet cell metaplasia/hyperplasia and mucus formation, MUC5AC synthesis, loss of tight junction proteins, and increased expression of Th2 cytokines/transcription factors. In addition, SHIV and CS synergistically reduced the lung function and increased the extrathoracic tracheal ring thickness. Interestingly, SHIV-infection generated significant numbers of HIV-gp120+ epithelial cells (HGECs) in small airways and alveoli and their numbers doubled in CS+SHIV-infected lungs. We conclude that even with cART, SHIV independently induces CB and pro-COPD changes in the lung and the effects are exacerbated by CS.

Authors

Hitendra S. Chand, Rodrigo Vazquez-Guillamet, Christopher M. Royer, Karin Rudolph, Neerad C. Mishra, Shashi P. Singh, Shah S. Hussain, Edward G. Barrett, Shannon Callen, Siddappa N. Byrareddy, Maria Cristina Vazquez Guillamet, Jawad Abukhalaf, Aryaz Sheybani, Vernat Exil, Veena Raizada, Hemant Agarwal, Madhavan Nair, Francois Villinger, Shilpa Buch, Mohan Sopori

CD84 regulates PD-1/PD-L1 expression and function in chronic lymphocytic leukemia

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Abstract

Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature, B lymphocytes in the peripheral blood, lymphoid tissues and bone marrow. CLL is characterized by profound immune defects, leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been implicated as playing an important role in anti-tumor responses. The CLL-mediated T cell exhaustion is achieved by aberrant expression of several inhibitory molecules on CLL and their environment, prominently the PD-L1/PD-1 receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that a cell-cell interaction mediated through human and mouse CD84 upregulates PDL1 expression on CLL and their microenvironment, and PD1 expression on T cells. This resulted in suppression of T cell response and activity in vitro and in vivo. Thus, our results demonstrated a role for CD84 in regulation of immune checkpoints by leukemia cells, and suggested CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.

Authors

Hadas Lewinsky, Avital F. Barak, Victoria Huber, Matthias P. Kramer, Lihi Radomir, Lital Sever, Irit Orr, Vita Mirkin, Nili Dezorella, Mika Shapiro, Yosef Cohen, Lev Shvidel, Martina Seiffert, Yair Herishanu, Shirly Becker-Herman, Idit Shachar