Activating innate immunity in cancer cells through cytoplasmic nucleic acid sensing pathways, a phenomenon known as “viral mimicry,” has emerged as an effective strategy to convert immunologically “cold” tumors into “hot.” Through a curated CRISPR-based screen of RNA helicases, we identified DExD/H-box helicase 9 (DHX9) as a potent repressor of double-stranded RNA (dsRNA) in small cell lung cancers (SCLC). Depletion of DHX9 induced accumulation of cytoplasmic dsRNA and triggered tumor-intrinsic innate immunity. Intriguingly, ablating DHX9 also induced aberrant accumulation of R-loops, which resulted in an increase of DNA damage–derived cytoplasmic DNA and replication stress in SCLCs. In vivo, DHX9 deletion promoted a decrease in tumor growth while inducing a more immunogenic tumor microenvironment, invigorating responsiveness to immune-checkpoint blockade. These findings suggest that DHX9 is a crucial repressor of tumor-intrinsic innate immunity and replication stress, representing a promising target for SCLC and other “cold” tumors in which genomic instability contributes to pathology. Significance: One promising strategy to trigger an immune response within tumors and enhance immunotherapy efficacy is by inducing endogenous “virus-mimetic” nucleic acid accumulation. Here, we identify DHX9 as a viral-mimicry-inducing factor involved in the suppression of double-stranded RNAs and R-loops and propose DHX9 as a novel target to enhance antitumor immunity. See related commentary by Chiappinelli, p. 389. This article is featured in Selected Articles from This Issue, p. 384

Phenotypic alterations in the lung epithelium have been widely implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but the precise mechanisms orchestrating this persistent inflammatory process remain unknown because of the complexity of lung parenchymal and mesenchymal architecture. To identify cell type–specific mechanisms and cell–cell interactions among the multiple lung resident cell types and inflammatory cells that contribute to COPD progression, we profiled 57,918 cells from lungs of patients with COPD, smokers without COPD, and never-smokers using single-cell RNA sequencing technology. We predicted pseudotime of cell differentiation and cell-to-cell interaction networks in COPD. Although epithelial components in never-smokers were relatively uniform, smoker groups represent extensive heterogeneity in epithelial cells, particularly in alveolar type 2 (AT2) clusters. Among AT2 cells, which are generally regarded as alveolar progenitors, we identified a unique subset that increased in patients with COPD and specifically expressed a series of chemokines including CXCL1 and CXCL8. A trajectory analysis revealed that the inflammatory AT2 cell subpopulation followed a unique differentiation path, and a prediction model of cell-to-cell interactions inferred significantly increased intercellular networks of inflammatory AT2 cells. Our results identify previously unidentified cell subsets and provide an insight into the biological and clinical characteristics of COPD pathogenesis.

Extracellular vesicles (EVs), including exosomes, are recognized as promising functional targets involved in disease mechanisms. However, the intravital heterogeneity of EVs remains unclear, and the general limitation for analyzing EVs is the need for a certain volume of biofluids. Here, we present cellulose nanofiber (CNF) sheets to resolve these issues. We show that CNF sheets capture and preserve EVs from ~10 μL of biofluid and enable the analysis of bioactive molecules inside EVs. By attaching CNF sheets to moistened organs, we collect EVs in trace amounts of ascites, which is sufficient to perform small RNA sequence analyses. In an ovarian cancer mouse model, we demonstrate that CNF sheets enable the detection of cancer-associated miRNAs from the very early phase when mice did not have apparent ascites, and that EVs from different locations have unique miRNA profiles. By performing CNF sheet analyses in patients, we identify further location-based differences in EV miRNA profiles, with profiles reflecting disease conditions. We conduct spatial exosome analyses using CNF sheets to reveal that ascites EVs from cancer patients exhibit location-dependent heterogeneity. This technique could provide insights into EV biology and suggests a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning.

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. The frequency of DCIS is increasing because of routine mammography; however, the biological features and intratumoral heterogeneity of DCIS remain obscure. To address this deficiency, we performed single-cell transcriptomic profiling of DCIS and invasive ductal carcinoma (IDC). DCIS was found to be composed of several transcriptionally distinct subpopulations of cancer cells with specific functions. Several transcripts, including long noncoding RNAs, were highly expressed in IDC compared with DCIS and might be related to the invasive phenotype. Closeness centrality analysis revealed extensive heterogeneity in DCIS, and the prediction model for cell-to-cell interactions implied that the interaction network among luminal cells and immune cells in DCIS was comparable with that in IDC. In addition, transcriptomic profiling of HER2+ luminal DCIS indicated HER2 genomic amplification at the DCIS stage. These data provide novel insight into the intratumoral heterogeneity and molecular features of DCIS, which exhibit properties similar to IDC. Significance: Investigation of the molecular features of ductal carcinoma in situ at single cell resolution provides new insights into breast cancer biology and identifies candidate therapeutic targets and diagnostic biomarkers.

Rho GTPase Rac1 is a central regulator of F‐actin organization and signal transduction to control plasma membrane dynamics and cell proliferation. Dysregulated Rac1 activity is often observed in various cancers including breast cancer and is suggested to be critical for malignancy. Here, we showed that the ubiquitin E3 ligase complex Cullin‐3 (CUL3)/KCTD10 is essential for epidermal growth factor (EGF)‐induced/human epidermal growth factor receptor 2 (HER2)‐dependent Rac1 activation in HER2‐positive breast cancer cells. EGF‐induced dorsal membrane ruffle formation and cell proliferation that depends on both Rac1 and HER2 were suppressed in CUL3‐ or KCTD10‐depleted cells. Mechanistically, CUL3/KCTD10 ubiquitinated RhoB for degradation, another Rho GTPase that inhibits Rac1 activation at the plasma membrane by suppressing endosome‐to‐plasma membrane traffic of Rac1. In HER2‐positive breast cancers, high expression of Rac1 mRNA significantly correlated with poor prognosis of the patients. This study shows that this novel molecular axis (CUL3/KCTD10/RhoB) positively regulates the activity of Rac1 in HER2‐positive breast cancers, and our findings may lead to new treatment options for HER2‐ and Rac1‐positive breast cancers.

Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation.