Oncologies
Abstract
Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared to matched non-invasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified three distinct transcriptomic groups in invasive organoids, two of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGFB1, IL6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify unique molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.
Authors
Yea Ji Jeong, Hildur Knutsdottir, Fatemeh Shojaeian, Michael G. Lerner, Maria F. Wissler, Elodie Henriet, Tammy Ng, Shalini Datta, Bernat Navarro-Serer, Peter Chianchiano, Benedict Kinny-Köster, Jacquelyn W. Zimmerman, Genevieve Stein-O'Brien, Matthias M. Gaida, James R. Eshleman, Ming-Tseh Lin, Elana J. Fertig, Andrew J. Ewald, Joel S. Bader, Laura D. Wood
Abstract
Emerging evidence suggests that cryptic translation within long noncoding RNAs (lncRNAs) may produce novel proteins with important developmental/physiological functions. However, the role of this cryptic translation in complex diseases (e.g., cancer) remains elusive. Here, we applied an integrative strategy combining ribosome profiling and CRISPR/Cas9 screening with large-scale analysis of molecular/clinical data for breast cancer (BC) and identified estrogen receptor α–positive (ER+) BC dependency on the cryptic ORFs encoded by lncRNA genes that were upregulated in luminal tumors. We confirmed the in vivo tumor-promoting function of an unannotated protein, GATA3-interacting cryptic protein (GT3-INCP) encoded by LINC00992, the expression of which was associated with poor prognosis in luminal tumors. GTE-INCP was upregulated by estrogen/ER and regulated estrogen-dependent cell growth. Mechanistically, GT3-INCP interacted with GATA3, a master transcription factor key to mammary gland development/BC cell proliferation, and coregulated a gene expression program that involved many BC susceptibility/risk genes and impacted estrogen response/cell proliferation. GT3-INCP/GATA3 bound to common cis regulatory elements and upregulated the expression of the tumor-promoting and estrogen-regulated BC susceptibility/risk genes MYB and PDZK1. Our study indicates that cryptic lncRNA-encoded proteins can be an important integrated component of the master transcriptional regulatory network driving aberrant transcription in cancer, and suggests that the “hidden” lncRNA-encoded proteome might be a new space for therapeutic target discovery.
Authors
Caishang Zheng, Yanjun Wei, Peng Zhang, Longyong Xu, Zhenzhen Zhang, Kangyu Lin, Jiakai Hou, Xiangdong Lv, Yao Ding, Yulun Chiu, Antrix Jain, Nelufa Islam, Anna Malovannaya, Yun Wu, Feng Ding, Han Xu, Ming Sun, Xi Chen, Yiwen Chen
Abstract
Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.
Authors
Saket Jain, Jonathan W. Rick, Rushikesh S. Joshi, Angad Beniwal, Jordan Spatz, Sabraj Gill, Alexander Chih-Chieh Chang, Nikita Choudhary, Alan T. Nguyen, Sweta Sudhir, Eric J. Chalif, Jia-Shu Chen, Ankush Chandra, Alexander F. Haddad, Harsh Wadhwa, Sumedh S. Shah, Serah Choi, Josie L. Hayes, Lin Wang, Garima Yagnik, Joseph F. Costello, Aaron Diaz, Dieter Henrik Heiland, Manish K. Aghi
Abstract
CD8+ exhausted T-cells (Tex) are heterogeneous. PD-1 inhibitors reinvigorate progenitor Tex, which subsequently differentiate into irresponsive terminal Tex. Maintaining durable proliferative capacity of progenitor Tex is important but remains unclear. Here, we showed that low-dose DNA demethylating agent decitabine-pretreated CD8+ progenitor Tex had enhanced proliferation and effector function against tumors after anti-PD-1 treatment in vitro. Decitabine-plus-anti-PD-1 treatment promoted the activation and expansion of tumor-infiltrated CD8+ progenitor Tex and efficiently suppressed tumor growth in multiple tumor models. Transcriptional and epigenetic profiling of tumor-infiltrated T cells demonstrated that decitabine-plus-anti-PD-1 combination markedly elevated the clonally expansion and cytolytic activity of progenitor Tex compared with anti-PD-1 monotherapy and restrained CD8+ T-cell terminal differentiation. Strikingly, decitabine-plus-anti-PD-1 sustained the expression and activity of AP-1 transcription factor JunD, which was reduced following PD-1 blockade therapy. Downregulation of JunD repressed T cell proliferation and activating JNK/AP-1 signaling in CD8+ T-cells enhanced the antitumor capacity of PD-1 inhibitors. Together, epigenetic agent remodels CD8+ progenitor Tex and improves responsiveness to anti-PD-1 therapy.
Authors
Xiang Li, Yaru Li, Liang Dong, Yixin Chang, Xingying Zhang, Chunmeng Wang, Meixia Chen, Xiaochen Bo, Hebing Chen, Weidong Han, Jing Nie
Abstract
Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcomes, yet the underlying mechanisms are incompletely understood. Here, we show that deleting c-Src abrogates the activity of Forkhead Box M1 (FOXM1), a master transcriptional regulator of the cell cycle, in a genetically engineered model mimicking the Luminal B molecular subtype of breast cancer. By phosphorylating it on two tyrosine residues, c-Src stimulates the nuclear localization of FOXM1 and the expression of its target genes, including key regulators of G2-M cell cycle progression as well as c-Src itself. This positive feedback loop drives proliferation in genetically engineered and patient-derived models of Luminal B-like breast cancer. Targeting this mechanism, including through novel compounds that destabilize the FOXM1 protein, induces G2-M cell cycle arrest and apoptosis, blocking tumor progression and impairing metastasis. We identify a positive correlation between FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcomes and associates with the Luminal B subtype, which responds poorly to approved therapies. These findings indicate that a regulatory network centered on c-Src and FOXM1 is a targetable vulnerability in aggressive luminal breast cancers.
Authors
Ipshita Nandi, Harvey W. Smith, Virginie Sanguin-Gendreau, Linjia Ji, Alain Pacis, Vasilios Papavasiliou, Dongmei Zuo, Stella Nam, Sherif S. Attalla, Sung Hoon Kim, Sierra Lusson, Hellen Kuasne, Anne-Marie Fortier, Paul Savage, Constanza Martinez Ramirez, Morag Park, John A. Katzenellenbogen, Benita S. Katzenellenbogen, William J. Muller
Abstract
Lysosomal inhibition elicited by palmitoyl protein transferase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC), but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12, was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naïve T cells, and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells, engendered adaptive immunity and tumor rejection in “immune hot” tumors but not in “immune cold” tumors. These findings demonstrate LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.
Authors
Monika Bhardwaj, Jennifer J. Lee, Amanda M. Versace, Sandra L. Harper, Aaron R. Goldman, Mary Ann S. Crissey, Vaibhav Jain, Mahendra Pal Singh, Megane Vernon, Andrew E. Aplin, Seokwoo Lee, Masao Morita, Jeffrey D. Winkler, Qin Liu, David W. Speicher, Ravi K. Amaravadi
Abstract
STING-Type I interferon (IFN) signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonist as monotherapy has shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that Protein phosphatase 2A (PP2A) with its specific B regulatory subunit STRN4 negatively regulated STING-Type I IFN in macrophages. Mice with macrophages PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that hippo kinase MST1/2 was required for STING activation. STING agonist induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human GBM patients, YAP/TAZ was highly expressed in tumor-associated macrophages but not in non-tumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ is a previously unappreciated mechanism that mediate immunosuppression in tumor-associated macrophages and targeting PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy.
Authors
Winson S. Ho, Isha Mondal, Beisi Xu, Oishika Das, Raymond C. Sun, Pochin Chiou, Xiaomin Cai, Foozhan Tahmasebinia, Caren Yu-Ju Wu, Zhihao Wu, William Matsui, Michael Lim, Zhipeng Meng, Rongze Olivia Lu
Abstract
Inflammatory mediators released by cancer cells promote the induction of immune suppression and tolerance in myeloid cells. Interleukin-1 receptor-associated kinase-3 (IRAK3) is a pseudokinase that inhibits IL-1/TLR signaling but its role in patients treated with immune checkpoint blockade (ICB) therapy remains unclear. Using RNAseq data from the IMvigor210 trial, we found that tumors with high IRAK3 expressions showed enriched anti-inflammatory pathways and worse clinical response to ICB therapy. Upon IRAK3 protein deletion with CRISPR/Cas9, primary human monocytes displayed altered global protein expression and phosphorylation in quantitative proteomics and released more pro-inflammatory cytokines in response to stimulation. Bone-marrow derived macrophages from an IRAK3 CRISPR knockout (KO) mouse model demonstrated a pro-inflammatory phenotype and enhanced sensitivity to TLR agonists, compared to wild-type cells. IRAK3 deficiency delayed the growth of carcinogen-induced and oncogene-driven murine cancer cells and induced enhanced activation in myeloid cells and T cells. Upon ICB treatment, IRAK3 KO mice showed enrichment of TCF1+PD-1+ stem-like memory CD8+ T cells and resulted in superior growth inhibition of immunologically cold tumors in vivo. Altogether, our study demonstrated a novel cancer-driven immune tolerance program controlled by IRAK3 in humans and mice and proposed its suitability as an immunotherapy target.
Authors
Gürcan Tunalı, Marta Rúbies Bedós, Divya Nagarajan, Patrik Fridh, Irineos Papakyriacou, Yumeng Mao
Abstract
Hypersecretory malignant cells underlie therapeutic resistance, metastasis, and poor clinical outcomes. However, the molecular basis for malignant hypersecretion remains obscure. Here, we showed that epithelial-to-mesenchymal transition (EMT) initiates exocytic and endocytic vesicular trafficking programs in lung cancer. The EMT-activating transcription factor ZEB1 executed a PI4KIIIβ-to-PI4KIIα (PI4K2A)-dependency switch that drove PI4P synthesis in Golgi and endosomes. EMT enhanced the vulnerability of lung cancer cells to PI4K2A small molecule antagonists. PI4K2A formed a MYOIIA-containing protein complex that facilitated secretory vesicle biogenesis in the Golgi, thereby establishing a hypersecretory state involving osteopontin (SPP1) and other pro-metastatic ligands. In the endosomal compartment, PI4K2A accelerated recycling of SPP1 receptors to complete an SPP1-dependent autocrine loop and interacted with HSP90 to prevent lysosomal degradation of AXL receptor tyrosine kinase, a driver of cell migration. These results show that EMT coordinates exocytic and endocytic vesicular trafficking to establish a therapeutically actionable hypersecretory state that drives lung cancer progression.
Authors
Xiaochao Tan, Guan-Yu Xiao, Shike Wang, Lei Shi, Yanbin Zhao, Xin Liu, Jiang Yu, William K. Russell, Chad J. Creighton, Jonathan M. Kurie
Abstract
Tumor suppressor TP53 is the most frequently mutated gene in human cancers. Mutant p53 (mutp53) proteins often accumulate to very high levels in human cancers to promote cancer progression through the gain-of-function (GOF) mechanism. Currently, the mechanism underlying mutp53 accumulation and GOF is incompletely understood. Here, we identified TRIM21 as a critical E3 ubiquitin ligase of mutp53 by screening for specific mutp53-interacting proteins. TRIM21 directly interacted with mutp53 but not wild-type p53, resulting in ubiquitination and degradation of mutp53 to suppress mutp53 GOF in tumorigenesis. TRIM21 deficiency in cancer cells promoted mutp53 accumulation and GOF in tumorigenesis. Compared with p53R172H knock-in mice, which displayed mutp53 accumulation specifically in tumors but not normal tissues, TRIM21 deletion in p53R172H knock-in mice resulted in mutp53 accumulation in normal tissues, an earlier tumor onset, and a shortened lifespan of mice. Furthermore, TRIM21 was frequently downregulated in some human cancers, including colorectal and breast cancers, and low TRIM21 expression was associated with poor prognosis in patients with cancers carrying mutp53. Our results revealed a critical mechanism underlying mutp53 accumulation in cancers, and also uncovered an important tumor-suppressive function of TRIM21 and its mechanism in cancers carrying mutp53.
Authors
Juan Liu, Cen Zhang, Dandan Xu, Tianliang Zhang, Chun-Yuan Chang, Jianming Wang, Jie Liu, Lanjing Zhang, Bruce G. Haffty, Wei-Xing Zong, Wenwei Hu, Zhaohui Feng
Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)