Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 ...blockade. Additionally, elevated collagen correlates with decreased total CD8
T cells and increased exhausted CD8
T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations.
Increased expression of zinc finger E-box binding homeobox 1 (ZEB1) is associated with tumor grade and metastasis in lung cancer, likely due to its role as a transcription factor in ...epithelial-to-mesenchymal transition (EMT). Here, we modeled malignant transformation in human bronchial epithelial cells (HBECs) and determined that EMT and ZEB1 expression are early, critical events in lung cancer pathogenesis. Specific oncogenic mutations in TP53 and KRAS were required for HBECs to engage EMT machinery in response to microenvironmental (serum/TGF-β) or oncogenetic (MYC) factors. Both TGF-β- and MYC-induced EMT required ZEB1, but engaged distinct TGF-β-dependent and vitamin D receptor-dependent (VDR-dependent) pathways, respectively. Functionally, we found that ZEB1 causally promotes malignant progression of HBECs and tumorigenicity, invasion, and metastases in non-small cell lung cancer (NSCLC) lines. Mechanistically, ZEB1 expression in HBECs directly repressed epithelial splicing regulatory protein 1 (ESRP1), leading to increased expression of a mesenchymal splice variant of CD44 and a more invasive phenotype. In addition, ZEB1 expression in early stage IB primary NSCLC correlated with tumor-node-metastasis stage. These findings indicate that ZEB1-induced EMT and associated molecular changes in ESRP1 and CD44 contribute to early pathogenesis and metastatic potential in established lung cancer. Moreover, TGF-β and VDR signaling and CD44 splicing pathways associated with ZEB1 are potential EMT chemoprevention and therapeutic targets in NSCLC.
RNA interference (RNAi) represents a promising strategy for identification and validation of putative therapeutic targets and for treatment of a myriad of important human diseases including cancer. ...However, the effective systemic in vivo delivery of small interfering RNA (siRNA) to tumors remains a formidable challenge. Using a robust self-assembly strategy, we develop a unique nanoparticle (NP) platform composed of a solid polymer/cationic lipid hybrid core and a lipid-poly(ethylene glycol) (lipid-PEG) shell for systemic siRNA delivery. The new generation lipidâpolymer hybrid NPs are small and uniform, and can efficiently encapsulate siRNA and control its sustained release. They exhibit long blood circulation ( t â/â â¼8 h), high tumor accumulation, effective gene silencing, and negligible in vivo side effects. With this RNAi NP, we delineate and validate the therapeutic role of Prohibitin1 (PHB1), a target protein that has not been systemically evaluated in vivo due to the lack of specific and effective inhibitors, in treating non-small cell lung cancer (NSCLC) as evidenced by the drastic inhibition of tumor growth upon PHB1 silencing. Human tissue microarray analysis also reveals that high PHB1 tumor expression is associated with poorer overall survival in patients with NSCLC, further suggesting PHB1 as a therapeutic target. We expect this long-circulating RNAi NP platform to be of high interest for validating potential cancer targets in vivo and for the development of new cancer therapies.
Significance This study developed a new generation lipidâpolymer hybrid nanoparticle platform for effective systemic delivery of small interfering RNA (siRNA) to tumors, which represents a challenging hurdle for the widespread application of RNA interference (RNAi) in cancer research and therapy. With promising in vivo features such as long blood circulation, high tumor accumulation, and effective gene silencing, the hybrid siRNA nanoparticles were successfully used to reveal and validate a putative therapeutic target, Prohibitin1 (PHB1), in non-small cell lung cancer treatment. In vivo antitumor efficacy results and human tissue microarray analysis further suggested the feasibility of utilizing PHB1 siRNA nanoparticles as a novel therapeutic agent. This hybrid RNAi nanoparticle platform may serve as a valuable tool for validating potential cancer targets and developing new cancer therapies.
With increasing use of immunotherapy agents, pretreatment strategies for identifying responders and non-responders is useful for appropriate treatment assignment. We hypothesize that the local immune ...micro-environment of NSCLC is associated with patient outcomes and that these local immune features exhibit distinct radiologic characteristics discernible by quantitative imaging metrics. We assembled two cohorts of NSCLC patients treated with definitive surgical resection and extracted quantitative parameters from pretreatment CT imaging. The excised primary tumors were then quantified for percent tumor PDL1 expression and density of tumor-infiltrating lymphocyte (via CD3 count) utilizing immunohistochemistry and automated cell counting. Associating these pretreatment radiomics parameters with tumor immune parameters, we developed an immune pathology-informed model (IPIM) that separated patients into 4 clusters (designated A-D) utilizing 4 radiomics features. The IPIM designation was significantly associated with overall survival in both training (5 year OS: 61%, 41%, 50%, and 91%, for clusters A-D, respectively, P = 0.04) and validation (5 year OS: 55%, 72%, 75%, and 86%, for clusters A-D, respectively, P = 0.002) cohorts and immune pathology (all P < 0.05). Specifically, we identified a favorable outcome group characterized by low CT intensity and high heterogeneity that exhibited low PDL1 and high CD3 infiltration, suggestive of a favorable immune activated state. We have developed a NSCLC radiomics signature based on the immune micro-environment and patient outcomes. This manuscript demonstrates model creation and validation in independent cohorts.
The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines ...revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2′-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides.
Prospectively identifying who will benefit from adjuvant chemotherapy (ACT) would improve clinical decisions for non-small cell lung cancer (NSCLC) patients. In this study, we aim to develop and ...validate a functional gene set that predicts the clinical benefits of ACT in NSCLC.
An 18-hub-gene prognosis signature was developed through a systems biology approach, and its prognostic value was evaluated in six independent cohorts. The 18-hub-gene set was then integrated with genome-wide functional (RNAi) data and genetic aberration data to derive a 12-gene predictive signature for ACT benefits in NSCLC.
Using a cohort of 442 stage I to III NSCLC patients who underwent surgical resection, we identified an 18-hub-gene set that robustly predicted the prognosis of patients with adenocarcinoma in all validation datasets across four microarray platforms. The hub genes, identified through a purely data-driven approach, have significant biological implications in tumor pathogenesis, including NKX2-1, Aurora Kinase A, PRC1, CDKN3, MBIP, and RRM2. The 12-gene predictive signature was successfully validated in two independent datasets (n = 90 and 176). The predicted benefit group showed significant improvement in survival after ACT (UT Lung SPORE data: HR = 0.34, P = 0.017; JBR.10 clinical trial data: HR = 0.36, P = 0.038), whereas the predicted nonbenefit group showed no survival benefit for 2 datasets (HR = 0.80, P = 0.70; HR = 0.91, P = 0.82).
This is the first study to integrate genetic aberration, genome-wide RNAi data, and mRNA expression data to identify a functional gene set that predicts which resectable patients with non-small cell lung cancer will have a survival benefit with ACT.
Mutant KRAS (KM), the most common oncogene in lung cancer (LC), regulates fatty acid (FA) metabolism. However, the role of FA in LC tumorigenesis is still not sufficiently characterized. Here, we ...show that KMLC has a specific lipid profile, with high triacylglycerides and phosphatidylcholines (PC). We demonstrate that FASN, the rate-limiting enzyme in FA synthesis, while being dispensable in EGFR-mutant or wild-type KRAS LC, is required for the viability of KMLC cells. Integrating lipidomic, transcriptomic and functional analyses, we demonstrate that FASN provides saturated and monounsaturated FA to the Lands cycle, the process remodeling oxidized phospholipids, such as PC. Accordingly, blocking either FASN or the Lands cycle in KMLC, promotes ferroptosis, a reactive oxygen species (ROS)- and iron-dependent cell death, characterized by the intracellular accumulation of oxidation-prone PC. Our work indicates that KM dictates a dependency on newly synthesized FA to escape ferroptosis, establishing a targetable vulnerability in KMLC.
EZH2 overexpression promotes cancer by increasing histone methylation to silence tumor suppressor genes, but how EZH2 levels become elevated in cancer is not understood. In this study, we ...investigated the mechanisms by which EZH2 expression is regulated in non-small cell lung carcinoma cells by oncogenic KRAS. In cells harboring KRAS(G12C) and KRAS(G12D) mutations, EZH2 expression was modulated by MEK-ERK and PI3K/AKT signaling, respectively. Accordingly, MEK-ERK depletion decreased EZH2 expression in cells harboring the KRAS(G12C) mutation, whereas PI3K/AKT depletion decreased EZH2 expression, EZH2 phosphorylation, and STAT3 activity in KRAS(G12D)-mutant cell lines. Combined inhibition of EZH2 and MEK-ERK or PI3K/AKT increased the sensitivity of cells with specific KRAS mutations to MEK-ERK and PI3K/AKT-targeted therapies. Our work defines EZH2 as a downstream effector of KRAS signaling and offers a rationale for combining EZH2 inhibitory strategies with MEK-ERK- or PI3K/AKT-targeted therapies to treat lung cancer patients, as stratified into distinct treatment groups based on specific KRAS mutations.
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