Y320, initially identified as an immunomodulator, has been reported to reverse multidrug resistance to chemotherapeutic agents in breast and liver cancers. However, its antitumor activities and underlying mechanisms remain poorly elucidated. In this study, we first investigated the antitumor effects and mechanisms of Y320 in non-small cell lung cancer (NSCLC). Y320 exerted potent antitumor activities against NSCLC both in vitro and in vivo. It induced excessive autophagy, as evidenced by increased autophagosome and autolysosome formation, reduced sequestosome 1 (SQSTM1) protein levels, and conversion of microtubule-associated protein 1 light chain 3 beta (LC3B) from type I to type II. Concurrently, Y320 significantly triggered pyroptosis via the caspase-3/GSDME pathway, as indicated by cellular swelling, membrane blebbing, lactate dehydrogenase (LDH) release, increased propidium iodide (PI) uptake, and elevated levels of cleaved caspase-3 and GSDME-NT. Y320-induced pyroptosis depended on autophagy, and blockade of autophagy at the early stage reversed the occurrence of pyroptosis. Additionally, Y320 downregulated cellular FLICE-like inhibitory protein (c-FLIP) expression through the proteasomal pathway. Importantly, we were the first to verify that knockdown of c-FLIP induced pyroptosis via activation of the caspase-3/GSDME axis. Overexpression of c-FLIP reversed Y320-induced autophagy-dependent pyroptosis. Thus, Y320 induced excessive autophagy-dependent pyroptosis in NSCLC cells by downregulating c-FLIP, ultimately leading to cell death. In conclusion, our study identifies Y320 as a promising therapeutic candidate for NSCLC and elucidates that autophagy-dependent pyroptosis is involved in its antitumor mechanism.

Breast cancer (BC) metastasis remains a major cause of mortality, yet the molecular mechanisms driving this process are incompletely understood. This study identifies TMEM216, a transmembrane protein implicated in ciliary homeostasis, as a suppressor of lung metastasis in BC. Using mammary-specific Tmem216 knockout mice, we demonstrate that Tmem216 deficiency promotes lung metastasis without affecting primary tumor proliferation. Clinical analyses reveal reduced TMEM216 expression in metastatic lesions and aggressive cell lines, correlating with poor patient distant metastasis-free survival. Mechanistically, TMEM216 interacts with IGF1R and binds to IRS4 via the conserved K79-D1049 interaction, disrupting the IGF1R-IRS4 complex formation and suppressing IGF pathway activation. Rescue experiments in vitro and in vivo confirm that TMEM216-mediated metastasis inhibition depends on IGF signaling modulation. Tissue microarray analyses further establish an inverse correlation between TMEM216 levels and IGF1R phosphorylation in BC patients, with low TMEM216 expression associated with advanced metastasis. These findings delineate TMEM216 as a critical regulator of the IGF1R-IRS4 axis, offering therapeutic opportunities for targeting metastatic BC.

BACKGROUND:Autophagy is an evolutionarily conserved cellular process that maintains homeostasis. It enables tumor cells to survive and grow, which may lead to drug resistance. The PI3K/AKT/mTOR pathway is reported to play a key role in autophagy induction. BEX2 promotes cell growth and survival in cancer cells. However, its role in autophagy remains unclear. METHODS:Human non-small cell lung cancer (NSCLC) cell lines (A549, H1792 and H1299) and 293FT cells were used to investigate the function of BEX2 in autophagy. The experimental research was conducted via western blot, co-immunoprecipitation and confocal microscopy. RESULTS:Our data show that rapamycin induces BEX2 protein levels. Western blot and confocal microscopy analysis demonstrate that BEX2 regulates autophagy. Furthermore, BEX2 impairs PI3K/AKT/mTOR signaling. Specifically, BEX2 interacts with PIK3CA (PI3K catalytic subunit). BEX2 impairs the interaction of PIK3CA and p85 (PI3K regulatory subunit), therefore inhibiting PI3K activity. In summary, BEX2 regulates autophagy through the PI3K/AKT/mTOR signaling pathway by modulating the activity of PI3K, specifically dependent on the presence of PIK3CA. CONCLUSIONS:We discovered that BEX2 promotes autophagic flux via PI3K/AKT/mTOR signaling. BEX2 interacts with PIK3CA and impairs PIK3CA and p85 interaction, which hinders activation of PI3K/AKT/mTOR signaling and promotes autophagy induction.

Double-stranded RNAs (dsRNAs), known as conserved pathogen-associated molecular patterns, activate the integrated stress response via interferon-induced protein kinase R (PKR), leading to global translation inhibition. However, the interferon system is inactive in pluripotent cells, leaving the mechanisms of dsRNA sensing and translational control unclear. In this study, we utilized early zebrafish embryos as a model of pluripotent cells and discovered a PKR-independent blockage of translation initiation by dsRNA stimulation. Prkra dimer was identified as the genuine dsRNA sensor. Upon dsRNA binding, the dimerized dsRNA-binding domain 3 of Prkra becomes activated to sequester the eIF2 complexes from the translation machinery, inhibiting global protein synthesis. This distinctive embryonic stress response restricts RNA virus replication in zebrafish embryos, is conserved in mouse embryonic stem cells, and compensates PKR function in differentiated cells. Therefore, the Prkra-mediated dsRNA sensing and translation control may serve as a common strategy for cells to adapt to environmental stresses.

Nucleic acid sensing, the initial stage of pathogen detection, plays a fundamental role in innate immunity. Double-stranded RNAs, mainly of viral origin, are recognized by protein kinase R (PKR/Eif2ak2) in differentiated cells to trigger an integrated stress response (ISR), characterized by the inhibition of global translation. However, in pluripotent cells, the sensor of double-stranded RNAs in antiviral innate immunity is poorly defined and conflated with those in differentiated cells. In this study, we utilized early zebrafish embryos as a model for pluripotent cells and discovered that dsRNA stimulation induces cell necrosis and a PKR-independent blockage of translation initiation. We identified Prkra as the genuine dsRNA sensor in this unique embryonic ISR. Prkra binds to and polymerizes on dsRNAs, diverting the eIF2 complex away from the translation machinery. Consequently, the formation of the 43S preinitiation complex is significantly reduced, leading to a hindrance in global protein synthesis. This distinctive embryonic ISR not only restricts RNA virus SVCV replication in zebrafish embryos but also exhibits conservation in early mouse embryos and embryonic stem cells. Therefore, the Prkra-mediated translation blockage potentially represents a common antiviral strategy across species. ![Figure][1] ### Competing Interest Statement China patent 202310609693.3 has been authorized to MS, TL and AJC for the application of zebrafish embryos for dsRNA by-product detection. [1]: pending:yes

Primary cilia are enriched in signaling receptors, and defects in their formation or function can induce conditions such as polycystic kidney disease, postaxial hexadactyly, and microphthalmia. Mammalian Hedgehog (Hh) signaling is important in the development of primary cilia, and TMEM216, a transmembrane protein that localizes to the base of cilia, is also implicated in ciliogenesis in zebrafish. Here, we found that Tmem216 -deficient mice had impaired Hh signaling and displayed typical ciliopathic phenotypes. These phenomena were also observed in cells deficient in TMEM216. Furthermore, TMEM216 interacted with core Hh signaling proteins, including SUFU, a negative regulator of Hh, and GLI2/GLI3, transcription factors downstream of Hh. The competition between TMEM216 and SUFU for binding to GLI2/GLI3 inhibited the cleavage of GLI2/GLI3 into their repressor forms, which resulted in the nuclear accumulation of full-length GLI2 and the decreased nuclear localization of cleaved GLI3, ultimately leading to the activation of Hh signaling. Together, these data suggest that the TMEM216-SUFU-GLI2/GLI3 axis plays a role in TMEM216 deficiency–induced ciliopathies and Hh signaling abnormalities.

Three chromomycin derivatives, chromomycins A3 (1, CA3), A5 (2, CA5), and monodeacetylchromomycin A3 (3, MDA-CA3), were identified from the soil-derived Streptomyces sp. CGMCC 26516. A reinvestigation of the structure of CA5 is reported, of which the absolute configuration was unambiguously determined for the first time to be identical with that of CA3 based on nuclear magnetic resonance (NMR) data analysis as well as NMR and electronic circular dichroism calculations. Compounds 1–3 showed potent cytotoxicity against the non-small-cell lung cancer (NSCLC) cells (A549, H460, H157-c-FLIP, and H157-LacZ) and down-regulated the protein expression of c-FLIP in A549 cells. The IC50 values of chromomycins in H157-c-FLIP were higher than that in H157-LacZ. Furthermore, si-c-FLIP promoted anti-proliferation effect of chromomycins in NSCLC cells. In nude mice xenograft model, 1 and 2 both showed more potent inhibition on the growth of H157-lacZ xenografts than that of H157-c-FLIP xenografts. These results verify that c-FLIP mediates the anticancer effects of chromomycins in NSCLC.

The forkhead box M1 (FoxM1) protein, a transcription factor, plays critical roles in regulating tumor growth and drug resistance, while cellular FLICE-inhibitory protein (c-FLIP), an anti-apoptotic regulator, is involved in the ubiquitin–proteasome pathway. In this study, we investigated the effects of c-FLIP on the expression and ubiquitination levels of FoxM1 along with drug susceptibility in non-small-cell lung cancer (NSCLC) cells. We first showed that the expression levels of FoxM1 and c-FLIP were increased and positively correlated (R2 = 0.1106, P < 0.0001) in 90 NSCLC samples. The survival data from prognostic analysis demonstrated that high expression of c-FLIP and/or FoxM1 was related to poor prognosis in NSCLC patients and that the combination of FoxM1 and c-FLIP could be a more precise prognostic biomarker than either alone. Then, we explored the functions of c-FLIP/FoxM1 in drug resistance in NSCLC cell lines and a xenograft mouse model in vivo. We showed that c-FLIP stabilized FoxM1 by inhibiting its ubiquitination, thus upregulated the expression of FoxM1 at post-transcriptional level. In addition, a positive feedback loop composed of FoxM1, β-catenin and p65 also participated in c-FLIP–FoxM1 axis. We revealed that c-FLIP promoted the resistance of NSCLC cells to thiostrepton and osimertinib by upregulating FoxM1. Taken together, these results reveal a new mechanism by which c-FLIP regulates FoxM1 and the function of this interaction in the development of thiostrepton and osimertinib resistance. This study provides experimental evidence for the potential therapeutic benefit of targeting the c-FLIP–FoxM1 axis for lung cancer treatment.

Brain expressed X-linked gene 2 (BEX2) encoded protein was originally identified to promote transcription by interacting with several transcription factors in the DNA-binding complexes. Recently, BEX2 was found to be localized in cytosol and/or mitochondria and regulate apoptosis in cancer cells and tumor growth. However, the molecular mechanism underlying its roles in cancer cells remains unclear. Here, we report that crotonylated BEX2 plays an important role in inhibiting chemotherapeutic agent-induced apoptosis via enhancing mitophagy in human lung cancer cells. BEX2 promotes mitophagy by facilitating interaction between NDP52 and LC3B. Moreover, BEX2 crotonylation at K59 is critical in the BEX2-mediated mitophagy in lung cancer cells. The K59R mutation of BEX2 inhibits mitophagy by affecting the interaction of NDP52 and LC3B. BEX2 expression is elevated after anticancer drug treatment, and its overexpression inhibits chemotherapy-induced apoptosis. In addition, inhibition of BEX2-regulated mitophagy sensitizes tumor cells to apoptosis. Furthermore, BEX2 promotes tumor growth and inhibits apoptosis by regulating mitophagy in vivo. We also confirm that BEX2 is overexpressed in lung adenocarcinoma and is associated with poor prognosis in lymph node metastasis-free cancer. Therefore, combination treatment with pharmaceutical approaches targeting BEX2-induced mitophagy and anticancer drugs may represent a potential strategy for NSCLC therapy.

PDF file, 104K, Knock-down of integrin β1 expression significantly reduced p-ERK levels in M4E and PCI-37B cells.

PDF file, 58K, Over expression of MMP-2 in M4E-15 integrin beta 1 knock-down cells increased the invasive capability.

PDF file, 346K, DFS and OS analysis between patients with and without metastasis.

PDF file, 95K, DFS analysis with COX model.

PDF file, 359K, OS and DFS analysis of chemotherapy on all patients.

Distant metastasis remains the leading cause of high mortality in patients with non-small-cell lung cancer (NSCLC). DIRAS3 is a candidate tumor suppressor protein that is decreased in various tumors. However, the regulatory mechanism of DIRAS3 on metastasis of NSCLC remains unclear. Here, we found that DIRAS3 suppressed the migration of NSCLC cells. Besides, DIRAS3 stimulated the polyubiquitination of RAC1 and suppressed its protein expression. Furthermore, RNF19B, a member of the RBR E3 ubiquitin ligase family, was observed to be the E3 ligase involved in the DIRAS3-induced polyubiquitination of RAC1. DIRAS3 could promote the binding of RAC1 and RNF19B, thus enhancing the degradation of RAC1 by the ubiquitin-proteasome pathway. Finally, the DIRAS3-RNF19B-RAC1 axis was confirmed to be associated with the malignant progression of NSCLC. These findings may be beneficial for developing potential prognostic markers of NSCLC and may provide an effective treatment strategy.

PDF file, 216K, PS-341 induces ER stress response in NSCLC cells.

PDF file, 26K, Integrin β1 knock-down did not affected cell proliferation.

PDF file, 897K, IHC analyses on xenograft tumor tissues.

Abnormal activation of epidermal growth factor receptor (EGFR) promotes the development of Non-Small Cell Lung Cancer Cells (NSCLC). Chemoresistance to tyrosine kinase inhibitors (TKIs), which is elicited by EGFR mutations, is a key challenge for NSCLC treatment. In the present study, we demonstrate a critical role of gasdermin E (GSDME), an important protein for pyroptosis, in the maintenance of EGFR stability and activation. We found that GSDME depletion suppressed the EGFR-mediated proliferation of NSCLC cells in vitro. GSDME knockdown downregulated the protein level of CCND1 and inhibited the phosphorylation of ERK1/2 in NSCLC cells. Mechanistically, both GSDME-FL and GSDME-N fragment physically interacted with EGFR. GSDME interacted with cytoplasmic fragment (CT) of EGFR. GSDME knockdown inhibited EGFR dimerization and phosphorylation at tyrosine 1173 (EGFRY1173), which could activate ERK1/2. GSDME knockdown promoted EGFR degradation and phosphorylation at tyrosine 1045 (EGFRY1045). Importantly, GSDME-FL increased the stability of EGFR, while the GSDME-N fragment induced EGFR degradation. Together, our results demonstrate that the GSDME-EGFR interaction plays an important role in NSCLC development, reveal a previously unrecognized link between GSDME and EGFR stability and offer new insight into cancer pathogenesis.