BACKGROUND/AIM:Microsatellite-stable (MSS) colorectal cancer (CRC) generally responds poorly to immune checkpoint blockade, but some MSS tumors are T-cell rich. We examined whether such infiltration reflected effective immunity or functional immune constraint. CASE REPORT:A 77-year-old woman underwent resection of a mismatch repair-proficient (pMMR), MSS, low-mutational-burden CRC with a synchronous adenoma. Whole-exome sequencing of tumor, adenoma and adjacent normal tissue detected no shared high-confidence somatic mutations between tumor and adenoma within the sensitivity of this WES analysis and identified tumor-specific APC, KRAS and TP53 alterations. Tumor single-cell RNA sequencing yielded 7,569 cells, with T-lineage populations comprising 83.5%. Cytotoxic T cells showed cytolytic and dysfunction-associated features, regulatory T cells (Tregs) showed suppressive remodeling, and Th17 cells showed inflammatory/profibrotic programs. CellChat nominated stromal MIF/FN1-CD74/CD44 and extracellular-matrix communication with T-cell compartments. CONCLUSION:This molecular case report shows that T-cell abundance and immune effectiveness can be uncoupled in MSS CRC.

Genome-wide association studies (GWASs) have revealed the lung cancer susceptibility-associated non-coding SNP rs17728461 C/G. In this study, we demonstrated that rs17728461 is also associated with lung cancer outcome. The risk G allele increases the proliferative index and motility of cancer cells and promotes cancer metastasis in vivo in a xenograft mouse model. Mechanistically, rs17728461-G establishes a physical interchromosomal interaction between the rs17728461-bearing DNA fragment and the RAB27A gene locus and thereby increases RAB27A expression and promotes subsequent exosome secretion. eQTL analysis and immunostaining revealed an association between rs17728461-G and increased RAB27A expression in human lung cancers. These findings reveal a noncoding SNP-mediated interchromosomal regulatory mechanism underlying lung cancer progression.

Human carcinomas often gain aggressive characteristics and escape cell-type specific treatment regimens through cryptic shifts in lineage states. However, the underlying mechanisms that govern lineage plasticity in carcinomas are undefined. Here in this study, we found that PAX5, a neural/lymphatic transcription factor, contributed to neuroendocrine (NE) lineage transition. PAX5 was highly expressed in aggressive human NE carcinoma cells and tissues but not in non-NE cancer cells and tissues. Deletion of Pax5 in Rb1fl/fl;Trp53fl/fl mice caused a reduction of tumor vessels, loss of NE morphologic features and decreased expression of ASCL1, NCAM, and SYP, whereas ectopic expression of PAX5 in CC10-rtTA;TetO-hEGFRex19del/T790M mice adenocarcinomas and in LNCAP prostate cancer xenografts induces an angiogenic microenvironment and NE morphology. Importantly, antiangiogenic drugs reduced NE features of Rb1fl/fl;Trp53fl/fl tumors and blocked PAX5-induced NE transformation. These studies demonstrate an essential role of angiogenic microenvironment in transition/maintenance of NE lineage, suggesting that targeting PAX5 and its downstream signaling may modulate lineage transitions responsible for treatment failure in both SCNCs and adenocarcinomas.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype that currently lacks effective targeted therapies. Transcriptional co-regulator nuclear protein 1 (NUPR1) has been identified as a key stress-adaptive disordered protein that promotes tumor progression and therapy-induced resistance. In this study, we developed a robust high-throughput platform integrating in situ proximity ligation assay followed by DNA sequencing (isPLA-seq), NanoBiT assays, and C-degron degradation validation to screen for functional single-domain antibodies (sdAbs) targeting NUPR1. Consequently, sdAb#07.81 emerged as a lead candidate, demonstrating strong binding affinity and the ability to degrade endogenous NUPR1 in TNBC cells. Functional assays confirmed that sdAb#07.81 suppressed TNBC cell growth, induced premature senescence, and derepressed ferroptosis. In vivo validation using a 4T1 cell-derived fully immunocompetent murine model further established its therapeutic efficacy, with significant reductions in tumor size, NUPR1 expression, and cell proliferation. These findings highlight sdAb#07.81 as a promising therapeutic agent and validate the platform’s effectiveness for addressing intracellular disordered targets like NUPR1. This work underscores the potential of sdAbs as a cancer therapeutic and provides a foundation for advancing sdAb#07.81 into preclinical and clinical development to address the critical unmet needs of TNBC treatment.

IntroductionSmall cell lung cancer (SCLC) is characterized by significant heterogeneity and plasticity, contributing to its aggressive progression and therapy resistance. Autophagy, a conserved cellular process, is implicated in many cancers, but its role in SCLC remains unclear.MethodsUsing a genetically engineered mouse model (Rb1fl/fl; Trp53fl/fl; GFP-LC3-RFP-LC3△G), we tracked autophagic flux in vivo to investigate its effects on SCLC biology. Additional in vitro experiments were conducted to modulate autophagic flux in NE and non-NE SCLC cell lines.ResultsTumor subpopulations with high autophagic flux displayed increased proliferation, enhanced metastatic potential, and neuroendocrine (NE) characteristics. Conversely, low-autophagic flux subpopulations exhibited immune-related signals and non-NE traits. In vitro, increasing autophagy induced NE features in non-NE cell lines, while autophagy inhibition in NE cell lines promoted non-NE characteristics.DiscussionThis study provides a novel model for investigating autophagy in vivo and underscores its critical role in driving SCLC heterogeneity and plasticity, offering potential therapeutic insights.

Lung cancer is the leading cause of cancer death worldwide. 85 % of lung cancers are categorized by their histological types as a non-small cell lung cancer (NSCLC) subtype. While the MED23 subunit of the mediator complex has been implicated in lung cancer development, the precise underlying mechanism remains unclear. Our research indicates that elevated MED23 expression is linked to reduced overall survival rates in NSCLC. Depletion of MED23 triggers premature senescence in NSCLC cells. Furthermore, through co-IP and mass spectrometry analyses, we have identified BCLAF1 as a binding partner of MED23, with subsequent confirmation via PLA assays. Subsequently, NUPR1, a transcriptional cofactor known to induce premature senescence in lung cancer cells by disrupting autophagic processes, was validated as a downstream target of the MED23/BCLAF1 complex through RNA-seq and ChIP assays. Thus, the interaction between MED23 and BCLAF1 regulates NUPR1 expression, impacting autophagic flux and leading to premature senescence in NSCLC cells.

Neuroblastoma (NB) is one of the most common childhood malignancies. Sixty percent of patients present with widely disseminated clinical signs at diagnosis and exhibit poor outcomes. However, the molecular mechanisms triggering NB metastasis remain largely uncharacterized. In this study, we generated a transcriptomic atlas of 15 447 NB cells from eight NB samples, including paired samples of primary tumors and bone marrow metastases. We used time-resolved analysis to chart the evolutionary trajectory of NB cells from the primary tumor to the metastases in the same patient and identified a common ‘starter’ subpopulation that initiates tumor development and metastasis. The ‘starter’ population exhibited high expression levels of multiple cell cycle-related genes, indicating the important role of cell cycle upregulation in NB tumor progression. In addition, our evolutionary trajectory analysis demonstrated the involvement of partial epithelial-to-mesenchymal transition (p-EMT) along the metastatic route from the primary site to the bone marrow. Our study provides insights into the program driving NB metastasis and presents a signature of metastasis-initiating cells as an independent prognostic indicator and potential therapeutic target to inhibit the initiation of NB metastasis.

目的:筛选并验证gasdermin D(GSDMD)单域抗体(single-domain antibody,sdAb)及其生物学功能.方法:利用原位邻近连接分析(in situ proximity ligation assay,isPLA)结合高通量测序筛选抗GSDMD sdAbs候选序列;利用isPLA、Co-IP、GST pull down、等温滴定量热法(isothermal titration calorimetry,ITC)验证这些sdAbs与GSDMD的特异性结合;在脂多糖(LPS)和nigericin处理的细胞焦亡模型中,观察细胞表型变化;检测细胞上清液中白细胞介素 1-β(IL-1β)水平变化以及乳酸脱氢酶(LDH)的释放;通过Western印迹检测经上述处理后的细胞中GSDMD以及GSDMD N端结构域(GSDMD N terminus,GSDMD-NT)量的变化.结果:通过isPLA结合高通量测序方法筛选出GSDMD sdAb的候选序列,其中sdAb#26 与GSDMD C端结构域(GSDMD C terminus,GSDMD-CT)相互作用;与sdAb Con对照组相比,sdAb#26 处理高表达GSDMD细胞产生焦亡表型的细胞显著增多;细胞上清中释放的IL-1β以及LDH显著提高(t=68.54,P<0.001;t=5.909,P<0.01);GSDMD-NT产生量显著增加.结论:GSDMD sdAb具备操控GSDMD介导焦亡的潜力,为焦亡相关疾病的治疗提供了新思路.

As mediators of pyroptosis, gasdermins (GSDMs) are closely associated with systemic cytotoxicity or so-called side effects and are also involved in the inflammatory response during chemotherapy. Using in situ proximity ligation assay followed by sequencing (isPLA-seq), which we recently developed, we screened a single-domain antibody (sdAb) library and identified several sdAbs against Gasdermin E (GSDME) that specifically recognize the N-terminal domain (1-270 aa) of GSDME (GSDME-NT). One of them mitigated the release of inflammatory damage-associated molecular patterns (DAMPs) and cytokines, including high mobility group protein b1 (Hmgb1) and interleukin-1β (Il-1β), in isolated mouse alveolar epithelial cells (AECs) upon chemotherapeutic agent cis-diaminodichloroplatinum (CDDP) treatment. Further investigation showed that this anti-GSDME sdAb also alleviated CDDP-induced pyroptotic cell death and lung tissue injury and decreased systemic Hmgb1 release in C57/BL6 mice, due to GSDME inactivation. Collectively, our data define an inhibitory role of the specific sdAb against GSDME, providing a potential strategy for systemically alleviating chemotherapeutic toxicities in vivo.

A previous genome‐wide association study (GWAS) revealed an association of the noncoding SNP rs1663689 with susceptibility to lung cancer in the Chinese population. However, the underlying mechanism is unknown. In this study, using allele‐specific 4C‐seq in heterozygous lung cancer cells combined with epigenetic information from CRISPR/Cas9‐edited cell lines, we show that the rs1663689 C/C variant represses the expression of ADGRG6, a gene located on a separate chromosome, through an interchromosomal interaction of the rs1663689 bearing region with the ADGRG6 promoter. This reduces downstream cAMP‐PKA signaling and subsequently tumor growth both in vitro and in xenograft models. Using patient‐derived organoids, we show that rs1663689 T/T—but not C/C—bearing lung tumors are sensitive to the PKA inhibitor H89, potentially informing therapeutic strategies. Our study identifies a genetic variant‐mediated interchromosomal interaction underlying ADGRG6 regulation and suggests that targeting the cAMP‐PKA signaling pathway may be beneficial in lung cancer patients bearing the homozygous risk genotype at rs1663689.

Table S1. Oligonucleotide sequences used for RT-PCR, ChIP, 3C assays and luciferase reporters

<p>Table S3, related to Figure 4: List of Spi-B-dependent genes categorized by biological function.</p>

Table S2, related to Figure 4: Genes downregulated/upregulated >2-fold by Spi-B transient expression in A549 cells

<p>Figure S1, related to Figure 3. Effect of Spi-B on EMT. (A) Phase-contrast micrographs of HBECs expressing vector or Spi-B. Scale bars are 100μm; (B) western blot showed expression of Spi-B, E-cadherin, Vimentin and Actin; (C) Quantitative RT-PCR assay for Spi-B, Snail1, Snail2, Twist1 and ZEB1. Figure S2, related to Figure 3. (A) Phase-contrast micrographs of H526 cells expressing control or Spi-B shRNAs. Scale bars are 50μm; (B) BrdU in corporation assay of A549 expressing vector or Spi-B; (C) vector or Spi-B-expressing A549 cells were plated on fibronectin-coated plates. After 15 min, attached cells were counted. Scale bars represent 200 mm. Bar graph below shows number of adherent cells. Mean {plus minus} SD of three duplications is shown. Figure S3, related to Figure 5. Expression of claudin-2 in tumor-adjacent tissues. (A) IHC showed expression of claudin-2 in bronchial epithelial cells; (B) IHC showed expression of claudin-2 in alveolar epithelial cells (arrows). Scale bars are 50μm. Figure S4, related to Figure 6. Other Spi-B downregulated tight junction proteins, Claudin-14 and Crumbs3, are not able to rescue Spi-B induced cellular behavior. (A) A549 cells were transduced with Spi-B or Spi-B and Claudin-14 or Spi-B and Crumbs3 as indicated. Representative images of the clones in soft agar were shown in left panels. The bar chart shows the percentage of mass (blue), and loose colonies (red). Error bars indicate means {plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 100 μm. (B) A549 cells were transduced with Spi-B or Spi-B and Claudin-14 or Spi-B and Crumbs3 as indicated. Phase contrast of acini were shown in left panel and quantified in right panel. The bar chart shows the percentage of mass (blue) and grape-like (red). Error bars indicate means {plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 25 μm. (C) A549 cells were transduced with Spi-B or Spi-B and Claudin-14 or Spi-B and Crumbs3 as indicated and subjected to an invasion assay. Error bars indicate means{plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 100 μm. Figure S5. (A) A549 cells were transduced with Spi-B or Spi-B and shRNA of MMP9 as indicated and subjected to an invasion assay. (B) A549 cells were transduced with Spi-B or Spi-B and MMP9 inhibitor (TIMP1) as indicated and subjected to an invasion assay. Error bars indicate means{plus minus} SD for a representative experiment performed in triplicate. Scale bars represent 100 μm.</p>

Androgen receptor (AR) mutation is closely associated with prostate cancer (PCa) and is one of the mechanisms of resistance to PCa therapies such as AR antagonists. Although sequencing technologies like next-generation sequencing (NGS) contributes to the high-throughput and precise detection of AR mutations carried by PCa patients, the lack of interpretations of these clinical genetic variants has still been a roadblock for PCa-targeted precision medicine. Here, we established a designer yeast reporter assay to simulate natural androgen receptor (AR) selection using AR antagonists. Yeast HIS3 gene transactivation was associated with the ligand-induced recruitment of steroid receptor coactivator-1 (SRC-1) by AR mutants, where yeast growth in histidine-free me-dium was determined as the outcome. This assay is applicable to determine a wide range of clinical AR mutants including those with loss of function relating to androgen insensitivity syndrome (AIS), and those associated with PCa conferring resistance to AR antagonists such as enzalutamide (ENZ), bicalutamide (BIC), and cyproterone acetate (CPA). One clinical AR mutant previously reported to confer ENZ-resistance, F877L, was found to confer partial resistance to CPA as well using designer yeast. Our simple and efficient assay can enable precise one-pot screening of AR mutants, providing a reference for tailored medicine.

BACKGROUND:Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer without recognised morphologic or genetic heterogeneity. Based on the expression of four transcription factors, ASCL1, NEUROD1, POU2F3, and YAP1, SCLCs are classified into four subtypes. However, biological functions of these different subtypes are largely uncharacterised.METHODS:We studied intratumoural heterogeneity of resected human primary SCLC tissues using single-cell RNA-Seq. In addition, we undertook a series of in vitro and in vivo functional studies to reveal the distinct features of SCLC subtypes.RESULTS:We identify the coexistence of ASCL1+ and NEUROD1+ SCLC cells within the same human primary SCLC tissue. Compared with ASCL1+ SCLC cells, NEUROD1+ SCLC cells show reduced epithelial features and lack EPCAM expression. Thus, EPCAM can be considered as a cell surface marker to distinguish ASCL1+ SCLC cells from NEUROD1+ SCLC cells. We further demonstrate that NEUROD1+ SCLC cells exhibit higher metastatic capability than ASCL1+ SCLC cells and can be derived from ASCL1+ SCLC cells.CONCLUSIONS:Our studies unveil the biology and evolutionary trajectory of ASCL1+ and NEUROD1+ SCLC cells, shedding light on SCLC tumourigenesis and progression.

Cancer progression is associated with the evolutionary accumulation of genetic mutations that are biologically significant. Mutations of the androgen receptor (AR) are associated with the development of prostate cancer (PCa) by responding to non-androgenic hormones, and the lack of annotations in their responsiveness to hormone ligands remains a daunting challenge. Here, we have used a yeast reporter system to quickly evaluate the responsiveness of all fifty clinical AR mutations to a variety of steroidal ligands including dihydrotestosterone (DHT), 17β-estradiol (E2), progesterone (PROG), and cyproterone acetate (CPA). Based on an AR-driven reporter that synthesizes histidine, a basic amino acid required for yeast survival and propagation, the yeast reporter system enabling clonal selection was further empowered by combining with a random DNA mutagenesis library to simulate the natural evolution of AR gene under the selective pressures of steroidal ligands. In a time-frame of 1–2 weeks, 19 AR mutants were identified, in which 11 AR mutants were validated for activation by tested steroidal compounds. The high efficiency of our artificial evolution strategy was further evidenced by a sequential selection that enabled the discovery of multipoint AR mutations and evolution directions under the pressure of steroidal ligands. In summary, our designer yeast is a portable reporter module that can be readily adapted to streamline high-throughput AR-compound screening, used as a PCa clinical reference, and combined with additional bioassay systems to further extend its potential.

The switch from anchorage-dependent to anchorage-independent growth is essential for epithelial metastasis. The underlying mechanism, however, is not fully understood. In this study, we identified growth factor independent-1 (GFI1), a transcription factor that drives the transition from adherent endothelial cells to suspended hematopoietic cells during hematopoiesis, as a critical regulator of anchorage independence in lung cancer cells. GFI1 elevated the numbers of circulating and lung-infiltrating tumor cells in xenograft models and predicted poor prognosis of patients with lung cancer. Mechanistically, GFI1 inhibited the expression of multiple adhesion molecules and facilitated substrate detachment. Concomitantly, GFI1 reconfigured the chromatin structure of the RASGRP2 gene and increased its expression, causing Rap1 activation and subsequent sustained ERK activation upon detachment, and this led to ERK signaling dependency in tumor cells. Our studies unveiled a mechanism by which carcinoma cells hijacked a hematopoietic factor to gain anchorage independence and suggested that the intervention of ERK signaling may suppress metastasis and improve the therapeutic outcome of patients with GFI1-positive lung cancer.

N-degron pathways are a set of proteolytic systems that target the N-terminal destabilizing residues of substrates for proteasomal degradation. Recently, the Gly/N-degron pathway has been identified as a new branch of the N-degron pathway. The N-terminal glycine degron (Gly/N-degron) is recognized by ZYG11B and ZER1, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we present the crystal structures of ZYG11B and ZER1 bound to various Gly/N-degrons. The structures reveal that ZYG11B and ZER1 utilize their armadillo (ARM) repeats forming a deep and narrow cavity to engage mainly the first four residues of Gly/N-degrons. The α-amino group of the Gly/N-degron is accommodated in an acidic pocket by five conserved hydrogen bonds. These structures, together with biochemical studies, decipher the molecular basis for the specific recognition of the Gly/N-degron by ZYG11B and ZER1, providing key information for future structure-based chemical probe design.