One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I) that significantly promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non-ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced major histocompatibility class I (MHC-I)-mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient-derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation, empowers T-cell cytotoxicity, and thus elevates the tumor response to immunotherapy.
Hanchen Xu, Kevin Van der Jeught, Zhuolong Zhou, Lu Zhang, Tao Yu, Yifan Sun, Yujing Li, Changlin Wan, Kaman So, Degang Liu, Michael Frieden, Yuanzhang Fang, Amber L. Mosley, Xiaoming He, Xinna Zhang, George E. Sandusky, Yunlong Liu, Samy O. Meroueh, Chi Zhang, Aruna B. Wijeratne, Cheng Huang, Guang Ji, Xiongbin Lu
BACKGROUND. Current clinical management of patients with pulmonary nodules involves either repeated LDCT/CT scans or invasive procedures yet causes significant patient misclassification. An accurate non-invasive test is needed to identify malignant nodules and reduce unnecessary invasive tests. METHOD. We developed a diagnostic model based on targeted DNA methylation sequencing of 389 pulmonary nodule patients’ plasma samples, and then validated in 140 plasma samples independently. We tested the model in different stages and subtypes of pulmonary nodules. RESULTS. A 100-feature model was developed and validated for pulmonary nodule diagnosis: the model achieved a ROC-AUC of 0.843 on 140 independent validation samples with an accuracy of 0.800. The performance was well maintained in, 1) 6-20 mm size subgroup (N=100), with a sensitivity of 1.000 and adjusted NPV of 1.000 at 10% prevalence; 2) stage I malignancy (N=90), with a sensitivity of 0.971; 3) different nodule types - solid nodules (N=78) with a sensitivity of 1.000 and adjusted NPV of 1.000, part-solid nodules (N=75) with a sensitivity of 0.947 and adjusted NPV of 0.983, and ground-glass nodules (N=67) with a sensitivity of 0.964 and adjusted NPV of 0.989 at 10% prevalence. This methylation test, called PulmoSeek, outperformed PET-CT and two clinical prediction models (Mayo and Veterans Affairs) in discriminating malignant pulmonary nodules from benign ones. CONCLUSION. This study suggests that the blood-based DNA methylation model may provide a better test for classifying pulmonary nodules, which could help facilitate the accurate diagnosis of early-stage lung cancer from pulmonary nodule patients and guide clinical decisions. FUNDING. The National Key Research and Development Program of China; Science and Technology Planning Project of Guangdong Province; The National Natural Science Foundation of China National.
Wenhua Liang, Zhiwei Chen, Caichen Li, Jun Liu, Jinsheng Tao, Xin Liu, Dezhi Zhao, Weiqiang Yin, Hanzhang Chen, Chao Cheng, Fenglei Yu, Chunfang Zhang, Lunxu Liu, Hui Tian, Kaican Cai, Xiang Liu, Zheng Wang, Ning Xu, Qing Dong, Liang Chen, Yue Yang, Xiuyi Zhi, Hui Li, Xixiang Tu, Xiangrui Cai, Zeyu Jiang, Hua Ji, Lili Mo, Jiaxuan Wang, Jian-Bing Fan, Jianxing He
Glioblastoma (GBM) is composed of heterogeneous tumor cell populations, including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance in GBM, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by a reduction in cell proliferation and an increase in cell-cell adhesion and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of β-catenin at the cell surface, which suppressed Wnt/β-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin, a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation.
Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir
Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance, however, the mechanisms are not completely understood and thus therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and MDSCs, and increased CD8+ T-cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable anti-tumor immunity, was detected in mice administered a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a new strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.
Fan Huang, Christophe Goncalves, Margarita Bartish, Joelle Rémy-Sarrazin, Mark E. Issa, Brendan Cordeiro, Qianyu Guo, Audrey Emond, Mikhael Attias, William Yang, Dany Plourde, Jie Su, Marina Godoy Gimeno, Yao Zhan, Alba Galán, Tomasz Rzymski, Milena Mazan, Magdalena Masiejczyk, Jacek Faber, Elie Khoury, Alexandre Benoit, Natascha Gagnon, David Dankort, Fabrice Journe, Ghanem Ghanem, Connie M. Krawczyk, H. Uri Saragovi, Ciriaco A. Piccirillo, Nahum Sonenberg, Ivan Topisirovic, Christopher E. Rudd, Wilson H. Miller Jr., Sonia V. del Rincón
Although cancer cells are frequently faced with nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anti-cancer strategy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PCK1) was downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low glucose condition. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo UTP synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.
Jin Xiang, Chang Chen, Rui Liu, Dongmei Gou, Lei Chang, Haijun Deng, Qingzhu Gao, Wanjun Zhang, Lin Tuo, Xuanming Pan, Li Liang, Jie Xia, Luyi Huang, Ke Yao, Bohong Wang, Zeping Hu, Ailong Huang, Kai Wang, Ni Tang
SUMOylation emerged as the inducer for the sorting of bioactive molecules into extracellular vesicles (EVs) triggering lymphangiogenesis, further driving tumor lymph node (LN) metastasis, but the precise mechanisms remain largely unclear. Herein, we identified that bladder cancer (BCa) cell-secreted EVs mediated the intercellular communication with human lymphatic endothelial cells (HLECs) through the transmission of a long noncoding RNA ELNAT1, and promoted lymphangiogenesis and LN metastasis in a SUMOylation-dependent manner in both cultured BCa cell lines and mouse models. Mechanistically, ELNAT1 induced UBC9 overexpression to catalyze the SUMOylation of hnRNPA1 at lysine-113 residue, which mediated the recognition of ELNAT1 by endosomal sorting complex required for transport (ESCRT) and facilitated their packaging into EVs. EV-mediated ELNAT1 was specifically transmitted into HLECs and epigenetically activated SOX18 transcription to induce lymphangiogenesis. Importantly, blocking the SUMOylation of tumor by downregulating UBC9 expression markedly reduced lymphatic metastasis in EV-mediated ELNAT1-treated BCa in vivo. Clinically, EV-mediated ELNAT1 was correlated with LN metastasis and poor prognosis of patients with BCa. These findings highlight a molecular mechanism that EV-mediated ELNAT1/UBC9/SOX18 regulatory axis promotes the lymphangiogenesis and LN metastasis of BCa in a SUMOylation-dependent manner, and implicate ELNAT1 as an attractive therapeutic target for LN metastatic BCa.
Changhao Chen, Hanhao Zheng, Yuming Luo, Yao Kong, Mingjie An, Yuting Li, Wang He, Bowen Gao, Yue Zhao, Hao Huang, Jian Huang, Tianxin Lin
BACKGROUND. p16 positive oropharyngeal squamous cell carcinoma (OPSCC) patients are potentially cured with definitive treatment. However, there are currently no reliable biomarkers of treatment failure in p16 positive OPSCC. Pathologist-based visual assessment of tumor cell multinucleation has been shown to be independently prognostic of disease-free survival in p16 positive OPSCC. However, its quantification is time-intensive, subjective, and at risk of interobserver variability. METHODS. We present a deep learning-based metric, the multi-nucleation index (MuNI), for prognostication in p16 positive OPSCC. This approach quantifies tumor multi-nucleation from digitally scanned hematoxylin eosin (H&E)-stained slides. Representative H&E whole slide images from 1,094 previously untreated p16 positive OPSCC patients were acquired from six institutions for optimizing and validating MuNI. RESULTS. MuNI was prognostic for disease-free (DFS), overall (OS), or distant metastasis-free (DMFS) survival in p16 positive OPSCC with HRs of 1.78(95%CI:1.37-2.30), 1.94(1.44-2.60), and 1.88(1.43-2.47), respectively, independent of age, smoking status, treatment type, and T/N-categories in multivariable analyses. It was also prognostic for DFS, OS, and DMFS in OPSCC patients at stages I and III. CONCLUSION. MuNI holds promise as a low-cost, tissue non-destructive, H&E stain based digital biomarker test for counseling, treatment, and surveillance of p16 positive OPSCC patients. These data support further confirmation of MuNI in prospective trials. FUNDING. This work was supported by the National Cancer Institute of the National Institutes of Health (under award numbers 1U24CA199374-01, R01CA202752-01A, R01CA208236-01A1, R01CA216579-01A1, R01CA220581-01A1, 1U01CA239055-01), the National Institute for Biomedical Imaging and Bioengineering (1R43EB028736-01), the National Center for Research Resources (1C06RR12463-01), the VA Merit Review Award (IBX004121A) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DoD Breast Cancer Research Program Breakthrough Level 1 Award (W81XWH-19-1-0668), the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering, and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University, the Michael E. DeBakey VA Medical Center, an institutional pilot grant (1IK2CX001953) and Dan L Duncan Comprehensive Cancer Center Support Grant (NCI-CA125123). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the U.S. Department of Veterans Affairs, the Department of Defense, or the United States Government.
Can F. Koyuncu, Cheng Lu, Kaustav Bera, Zelin Zhang, Jun Xu, Paula Andrea Toro Castaño, Germán Corredor, Deborah Chute, Pingfu Fu, Wade L. Thorstad, Farhoud Faraji, Justin A. Bishop, Mitra Mehrad, Patricia D. Castro, Andrew G. Sikora, Lester D. R. Thompson, R. D. Chernock, Krystle A. Lang Kuhs, Jingqin Luo, Vlad C. Sandulache, David J. Adelstein, Shlomo Koyfman, James S. Lewis Jr., Anant Madabhushi
Approaches using a single type of data have been applied to classify human tumors. Here we integrate imaging features and transcriptomic data using a prospectively collected tumor bank. We demonstrate that increased maximum standardized uptake value on pretreatment 18F-fluorodeoxyglucose–positron emission tomography correlates with epithelial-to-mesenchymal transition (EMT) gene expression. We derived and validated 3 major molecular groups, namely squamous epithelial, squamous mesenchymal, and adenocarcinoma, using prospectively collected institutional (n = 67) and publicly available (n = 304) data sets. Patients with tumors of the squamous mesenchymal subtype showed inferior survival outcomes compared with the other 2 molecular groups. High mesenchymal gene expression in cervical cancer cells positively correlated with the capacity to form spheroids and with resistance to radiation. CaSki organoids were radiation-resistant but sensitive to the glycolysis inhibitor, 2-DG. These experiments provide a strategy for response prediction by integrating large data sets, and highlight the potential for metabolic therapy to influence EMT phenotypes in cervical cancer.
Jin Zhang, Ramachandran Rashmi, Matthew Inkman, Kay Jayachandran, Fiona Ruiz, Michael R. Waters, Perry W. Grigsby, Stephanie Markovina, Julie K. Schwarz
Prostate cancer (PCa) is the second leading cause of cancer death in American men. Androgen receptor (AR) signaling is essential for PCa cell growth/survival and remains a key therapeutic target for lethal castration-resistant PCa (CRPC). GATA2 is a pioneer transcription factor crucial for inducing AR expression/activation. We recently reported that MAPK4, an atypical MAPK, promotes tumor progression via noncanonical activation of AKT. Here, we demonstrated that MAPK4 activated AR by enhancing GATA2 transcriptional expression and stabilizing GATA2 protein through repression of GATA2 ubiquitination/degradation. MAPK4 expression correlated with AR activation in human CRPC. Concerted activation of both GATA2/AR and AKT by MAPK4 promoted PCa cell proliferation, anchorage-independent growth, xenograft growth, and castration resistance. Conversely, knockdown of MAPK4 decreased activation of both AR and AKT and inhibited PCa cell and xenograft growth, including castration-resistant growth. Both GATA2/AR and AKT activation were necessary for MAPK4 tumor-promoting activity. Interestingly, combined overexpression of GATA2 plus a constitutively activated AKT was sufficient to drive PCa growth and castration resistance, shedding light on an alternative, MAPK4-independent tumor-promoting pathway in human PCa. We concluded that MAPK4 promotes PCa growth and castration resistance by cooperating parallel pathways of activating GATA2/AR and AKT and that MAPK4 is a novel therapeutic target in PCa, especially CRPC.
Tao Shen, Wei Wang, Wolong Zhou, Ilsa Coleman, Qinbo Cai, Bingning Dong, Michael M. Ittmann, Chad J. Creighton, Yingnan Bian, Yanling Meng, David R. Rowley, Peter S. Nelson, David D. Moore, Feng Yang
Ovarian cancer (OC) is the most deadly gynaecological malignancy with unmet clinical need for new therapeutic approaches. The relaxin peptide is a pleiotropic hormone with reproductive functions in the ovary. Relaxin induces aggressive cell growth in several types of cancer, but the role of relaxin in OC is poorly understood. Here, we demonstrate that relaxin and its associated G-protein coupled receptor RXFP1 form an autocrine signaling loop essential for OC in vivo tumorigenesis, cell proliferation and viability. We have found that relaxin signaling activates expression of pro-oncogenic pathways including RHO, MAPK, Wnt, and Notch. We find that relaxin is detectable in OC tumors, ascites and serum. Further, inflammatory cytokines IL-6 and TNF-α activate transcription of relaxin via recruitment of STAT3 and NFκB to the proximal promoter initiating an autocrine feedback loop that potentiates expression. Inhibition of RXFP1 or relaxin increases cisplatin sensitivity of OC cell lines and abrogates in vivo tumor formation. Finally, we demonstrate that a relaxin neutralizing antibody reduces OC cell viability and sensitizes cells to cisplatin. Collectively, targeting relaxin-RXFP1 signaling offers a potential new therapeutic strategy for OC.
Helen E. Burston, Oliver A. Kent, Laudine Communal, Molly L. Udaskin, Ren X. Sun, Kevin R. Brown, Euihye Jung, Kyle E. Francis, Jose La Rose, Joshua K. Lowitz, Ronny Drapkin, Anne-Marie Mes-Masson, Robert Rottapel