A fibroblast-dependent TGFß1/sFRP2 noncanonical Wnt signaling axis promotes epithelial metaplasia in idiopathic pulmonary fibrosis.

Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGFß1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Biopsies from untreated patients showed higher fibroblast TGFß1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGFß1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzle-like receptor protein 2 (sFRP2), an unrecognized TGFß1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision cut lung slices (PCLS) from non-diseased donors, we found TGFß1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature Krt5+ basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGFß1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify TGFß1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.

SARS-CoV-2 infection of airway organoids reveals conserved use of Tetraspanin-8 by Ancestral, Delta, and Omicron variants.

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

Genetic and immunologic features of recurrent stage I lung adenocarcinoma.

Although surgery for early-stage lung cancer offers the best chance of cure, recurrence still occurs between 30 and 50% of the time. Why patients frequently recur after complete resection of early-stage lung cancer remains unclear. Using a large cohort of stage I lung adenocarcinoma patients, distinct genetic, genomic, epigenetic, and immunologic profiles of recurrent tumors were analyzed using a novel recurrence classifier. To characterize the tumor immune microenvironment of recurrent stage I tumors, unique tumor-infiltrating immune population markers were identified using single cell RNA-seq on a separate cohort of patients undergoing stage I lung adenocarcinoma resection and applied to a large study cohort using digital cytometry. Recurrent stage I lung adenocarcinomas demonstrated higher mutation and lower methylation burden than non-recurrent tumors, as well as widespread activation of known cancer and cell cycle pathways. Simultaneously, recurrent tumors displayed downregulation of immune response pathways including antigen presentation and Th1/Th2 activation. Recurrent tumors were depleted in adaptive immune populations, and depletion of adaptive immune populations and low cytolytic activity were prognostic of stage I recurrence. Genomic instability and impaired adaptive immune responses are key features of stage I lung adenocarcinoma immunosurveillance escape and recurrence after surgery.

SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator.

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.

Hedgehog Signaling in Lung Cancer: From Oncogenesis to Cancer Treatment Resistance.

Hedgehog signaling pathway is physiologically activated during embryogenesis, especially in lung development. It is also reactivated in many solid tumors. In lung cancer, Hedgehog pathway is closely associated with cancer stem cells (CSCs). Recent works have shown that CSCs produced a full-length Sonic Hedgehog (Shh) protein, with paracrine activity and induction of tumor development. Hedgehog pathway is also involved in tumor drug resistance in lung cancer, as cytotoxic chemotherapy, radiotherapy, and targeted therapies. This review proposes to describe the activation mechanisms of Hedgehog pathway in lung cancer, the clinical implications for overcoming drug resistance, and the perspectives for further research.

Spotlight on afatinib and its potential in the treatment of squamous cell lung cancer: the evidence so far.

Compared to adenocarcinoma, fewer effective treatment options are available for advanced or metastatic squamous cell carcinoma (SCC) of the lung. Afatinib is an orally administered, irreversible EGFR antagonist. As a second-generation tyrosine kinase inhibitor, it has been applied in the treatment of patients with EGFR-mutant non-small-cell lung cancer. Recently, several clinical trials have shown that afatinib leads to a significant improvement in progression-free survival and overall survival of patients with SCC. Moving forward, afatinib should be one of the options among tyrosine kinase inhibitors, monoclonal antibodies, and cytotoxicity chemotherapy drugs for SCC.

Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice.

Arteriovenous malformations (AVMs) are tortuous vessels characterized by arteriovenous (AV) shunts, which displace capillaries and shunt blood directly from artery to vein. Notch signaling regulates embryonic AV specification by promoting arterial, as opposed to venous, endothelial cell (EC) fate. To understand the essential role of endothelial Notch signaling in postnatal AV organization, we used inducible Cre-loxP recombination to delete Rbpj, a mediator of canonical Notch signaling, from postnatal ECs in mice. Deletion of endothelial Rbpj from birth resulted in features of AVMs by P14, including abnormal AV shunting and tortuous vessels in the brain, intestine and heart. We further analyzed brain AVMs, as they pose particular health risks. Consistent with AVM pathology, we found cerebral hemorrhage, hypoxia and necrosis, and neurological deficits. AV shunts originated from capillaries (and possibly venules), with the earliest detectable morphological abnormalities in AV connections by P8. Prior to AV shunt formation, alterations in EC gene expression were detected, including decreased Efnb2 and increased Pai1, which encodes a downstream effector of TGFß signaling. After AV shunts had formed, whole-mount immunostaining showed decreased Efnb2 and increased Ephb4 expression within AV shunts, suggesting that ECs were reprogrammed from arterial to venous identity. Deletion of Rbpj from adult ECs led to tortuosities in gastrointestinal, uterine and skin vascular beds, but had mild effects in the brain. Our results demonstrate a temporal requirement for Rbpj in postnatal ECs to maintain proper artery, capillary and vein organization and to prevent abnormal AV shunting and AVM pathogenesis.

The RasGAP proteins Ira2 and neurofibromin are negatively regulated by Gpb1 in yeast and ETEA in humans.

The neurofibromatosis type 1 (NF1) gene encodes the GTPase-activating protein (GAP) neurofibromin, which negatively regulates Ras activity. The yeast Saccharomyces cerevisiae has two neurofibromin homologs, Ira1 and Ira2. To understand how these proteins are regulated, we utilized an unbiased proteomics approach to identify Ira2 and neurofibromin binding partners. We demonstrate that the Gpb1/Krh2 protein binds and negatively regulates Ira2 by promoting its ubiquitin-dependent proteolysis. We extended our findings to show that in mammalian cells, the ETEA/UBXD8 protein directly interacts with and negatively regulates neurofibromin. ETEA contains both UBA and UBX domains. Overexpression of ETEA downregulates neurofibromin in human cells. Purified ETEA, but not a mutant of ETEA that lacks the UBX domain, ubiquitinates the neurofibromin GAP-related domain in vitro. Silencing of ETEA expression increases neurofibromin levels and downregulates Ras activity. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 (in yeast) and neurofibromin (in humans).

Activation of p53 by Dishevelled independent of Wnt or planar polarity pathways.

Dishevelled is a key component of the Wnt signaling and planar polarity pathways. We discovered that in selective cell types, it potently activates the transcriptional activity of the tumor suppressor p53. This action, however, is not dependent on the downstream of either the Wnt or the planar polarity pathways. Dishevelled signals to the first 50 amino acids of p53, which is the transactivation domain. The level of phosphorylation on several serine residues within that region of p53 increases in response to disheveled activation, partially contributing to p53 activation. The MAP kinase pathway and E1B55k may also be involved in this dishevelled-p53 connection. Our data provide evidence that there is a novel signaling pathway from Dishevelled to p53.