CD74-ROS1 L2026M mutant enhances autophagy through the MEK/ERK pathway to promote invasion, metastasis and crizotinib resistance in non-small cell lung cancer cells.

The treatment of non-small cell lung cancer (NSCLC) patients harboring a proto-oncogene tyrosine-protein kinase c-ros oncogene 1 (ROS1) fusion gene has greatly benefited from the use of crizotinib. However, drug resistance inevitably occurs after 1 year of treatment. Clinical studies have shown that patients with an L2026M mutation in the ROS1 kinase domain account for about 6% of the total number of crizotinib-resistant cases, which is an important group that cannot be ignored. To explore the mechanism involved, we constructed the HLA class II histocompatibility antigen gamma chain (CD74)-ROS1 L2026M mutant gene by fusion polymerase chain reaction (PCR) and transfected it into H460 and A549 cells. We found that the invasion and metastasis abilities of drug-resistant cells were increased. The results of monodansylcadaverine (MDC) staining, Acridine orange (AO) staining, and western blot indicated that the autophagy level of CD74-ROS1 L2026M mutant NSCLC cells was increased compared with the CD74-ROS1 group, and the inhibition of autophagy could reverse the increased invasion and metastasis abilities caused by the L2026M mutation. In addition, the L2026M mutation led to excessive activation of the MEK/ERK pathway, and MEK inhibitors could reduce the autophagy level, invasion, and metastasis abilities of cells; additionally, this process could be blocked by rapamycin, an activator of autophagy. Furthermore, crizotinib treatment activated expression of Src homology region 2 domain-containing phosphatase-2 (SHP2; also known as PTPN11) to upregulate the MEK/ERK pathway, and the combination of MEK inhibitors and crizotinib increased apoptosis compared with crizotinib alone. In conclusion, our results indicate that the MEK/ERK pathway mediates the induction of invasion, metastasis, and crizotinib resistance through autophagy caused by CD74-ROS1 L2026M mutation in NSCLC cells, and targeting MEK could reverse these processes.

Targeting HMGA1 contributes to immunotherapy in aggressive breast cancer while suppressing EMT.

Metastasis is an obstacle to the clinical treatment of aggressive breast cancer (BC). Studies have shown that high mobility group A1 (HMGA1) is abnormally expressed in various cancers and mediates tumor proliferation and metastasis. Here, we provided more evidence that HMGA1 mediated epithelial to mesenchymal transition (EMT) through the Wnt/ß-catenin pathway in aggressive BC. More importantly, HMGA1 knockdown enhanced antitumor immunity and improved the response to immune checkpoint blockade (ICB) therapy by upregulating programmed cell death ligand 1 (PD-L1) expression. Simultaneously, we revealed a novel mechanism by which HMGA1 and PD-L1 were regulated by the PD-L1/HMGA1/Wnt/ß-catenin negative feedback loop in aggressive BC. Taken together, we believe that HMGA1 can serve as a target for the dual role of anti-metastasis and enhancing immunotherapeutic responses.

ZYY-B-2, a novel ALK inhibitor, overcomes resistance to ceritinib by inhibiting P-gp function and induces apoptosis through mitochondrial pathway in ceritinib-resistant H2228 cells.

Targeting the Echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene is a promising therapeutic strategy for non-small-cell lung cancer (NSCLC) patients. With the advent of the first- and second-generation ALK inhibitors, the mortality rate of lung cancer has shown a downward trend, but almost inevitably, patients will eventually develop resistance, which severely limits the clinical application. Hence, developing new ALK inhibitors which can overcome resistance is essential. Here, we synthesized a novel ALK inhibitor 1-[4-[[5-Chloro-4-[[2-[(1-methylethyl)sulfonyl]phenyl]amino]-2-pyrimidinyl]amino]-3-methoxyphenyl]-3-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]-2-imidazolidinone (ZYY-B-2) based on the structure of the second-generation ALK inhibitor ceritinib. ZYY-B-2 exhibited impressive anti-proliferative effect in the EML4-ALK positive H2228 cells and ceritinib-resistant H2228 (H2228/Cer) cells. Meanwhile, ZYY-B-2 inhibited the activation of p-ALK in a concentration-dependent manner, and inactivated its downstream target proteins p-AKT and p-ERK to inhibit cell proliferation. Subsequently, we found that ZYY-B-2 blocked H2228 cells and H2228/Cer cells in G0/G1 phase and induced cells to undergo apoptosis through the mitochondrial pathway. The ability of its anti-proliferation and pro-apoptosis was significantly stronger than the second generation ALK inhibitor ceritinib. In addition, high expression of P-gp was found in H2228/Cer cells compared with H2228 cells. ZYY-B-2 could inhibit the expression of P-gp in a dose-dependent manner to overcome ceritinib resistance, and the suppression effect of ZYY-B-2 on P-gp might be related to its inhibition of PI3K/AKT signaling pathway. In summary, ZYY-B-2, a promising ALK inhibitor, shows potent activity against ceritinib-resistant cells, which provides experimental and theoretical basis for the further development of new ALK inhibitors.

Corrigendum: Exploring the pharmacological mechanisms of icaritin against nasopharyngeal carcinoma via network pharmacology and experimental validation.

[This corrects the article DOI: 10.3389/fphar.2022.993022.].

Construction of crizotinib resistant models with CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutations to explore resistance mechanism and treatment strategy.

Targeted therapy is an essential treatment for non-small cell lung cancer (NSCLC) that is always associated with the drug resistance. c-ros oncogene 1 (ROS1) gene point mutation is one of the leading factors causing drug resistance. However, the point mutation cell models of crizotinib are challenging to obtain, causing few reports on the drug resistance mechanism and the treatment strategy. We constructed CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant plasmids by fusion PCR technology and transfected them into A549 cells. Western blot and MTT assay proved that the drug-resistant cell lines were successfully transfected. The transwell assay confirmed that the mutant cells' motor abilities were significantly increased compared with the wild-type group. In addition, focal adhesion kinase (FAK) was significantly increased in mutant cells. Moreover, crizotinib resistance occurred in the mutant cells through the activation of FAK / phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) pathway. Next, crizotinib was combined with defactinib, a FAK inhibitor, to further explore its therapeutic effect. The results showed that the combination could significantly inhibit the proliferation, invasion and migration of mutant cells. In conclusion, we proved that CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant NSCLC cells were resistant to crizotinib through the activation of FAK/PI3K/AKT signaling pathway, and inhibiting FAK/PI3K/AKT signaling pathway activation by defactinib could overcome drug resistance in mutant cells.

FAT4 activation inhibits epithelial-mesenchymal transition (EMT) by promoting autophagy in H2228/Cer cells.

As a tumor suppressor in lung cancer, FAT atypical cadherin 4 (FAT4) has a critical role in epithelial-mesenchymal transition (EMT). However, the role of FAT4 in ceritinib-resistant anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC) EMT has not been reported. It is necessary to discuss the role of FAT4 in this process and its potential mechanism of interaction. We found that the expression level of FAT4 was downregulated markedly in ceritinib-resistant NCI-H2228 (H2228/Cer) cells. Jujuboside A, a FAT4 activator, diminished EMT and metastasis of H2228/Cer cells. Importantly, autophagy inhibition inverted the inhibitory effect of FAT4 activation on EMT. Furthermore, we found the regulatory action of FAT4 on autophagy was related to proteasome 26S subunit ubiquitin receptor and non-ATPase 4 (PSMD4) and proteasome 20S subunit beta 4 (PSMB4), and the inhibitory effect of autophagy on EMT might be related to ROS/NF-κB/IκB-α and Wnt/ß-catenin pathways. In conclusion, FAT4 activation can inhibit the process of EMT in H2228/Cer cells by promoting autophagy, which provides a potential target for ceritinib-resistant ALK positive NSCLC therapy.

Exploring the pharmacological mechanisms of icaritin against nasopharyngeal carcinoma via network pharmacology and experimental validation.

Background: Icaritin is a natural product with a wide range of anti-tumor effects. However, its anti-tumor mechanism has not been thoroughly studied. This study examined the inhibitory effect of icaritin on nasopharyngeal cancer and its underlying mechanism using network pharmacology along with in vivo and in vitro experiments. Methods: MTT and clone formation assays were used to detect the effects of icaritin on the viability and proliferation of nasopharyngeal carcinoma cells, followed by the construction of a HONE1 xenograft tumor model to evaluate the anti-tumor efficacy of icaritin in vivo. A public database was used to predict prospective targets, built a protein-protein interaction (PPI) network, and analyze gene enrichment and biological processes. Based on network pharmacological data, cell cycle-related proteins were identified using western blotting. Besides, cell cycle distribution, apoptosis, and intracellular reactive oxygen species (ROS) generation were identified using flow cytometry. In addition, SA-ß-Gal staining was performed to detect cellular senescence, and western blotting was performed to detect the expression of P53, P21, and other proteins to verify key signaling pathways. Results: Icaritin effectively inhibited the viability and proliferation of nasopharyngeal carcinoma cell lines and showed good anti-tumor activity against HONE1 nasopharyngeal carcinoma cells in vivo. Key protein targets, including AKT1, HSP90AA1, CDK4, CCND1, and EGFR, were screened using PPI network topology analysis. GO and KEGG analysis revealed that the cell cycle, p53 signaling, and cell senescence pathways may be the main regulatory pathways. Flow cytometry and western blot experiments showed that icaritin caused S-phase arrest and promoted an increase in ROS. SA-ß-Gal staining showed that icaritin significantly induced cellular senescence, and western blotting showed that the expression of senescence-related proteins p53 and P21 increased significantly. Moreover, inhibition of ROS levels by N-Acetylcysteine (NAC) enhanced cell viability, reversed cellular senescence and reduced cellular senescence-associated protein expression. Conclusion: The results of network pharmacological analysis and in vivo and in vitro experiments showed that icaritin effectively inhibited the growth of nasopharyngeal carcinoma cells, promoted ROS production, induced cellular senescence, and inhibited tumor cells, which are related to the regulation of P53/P21 signal pathway.

XS-2, a novel potent dual PI3K/mTOR inhibitor, exhibits high in vitro and in vivo anti-breast cancer activity and low toxicity with the potential to inhibit the invasion and migration of triple-negative breast cancer.

Breast cancer has become the most commonly diagnosed cancer, surpassing lung cancer, with 2.26 million new breast cancers worldwide in 2020. Hence, there is an urgent need to develop effective molecularly targeted therapeutic drugs to treat breast cancer. In this paper, we designed, synthesized and screened a novel thiophene-triazine derivative, XS-2, as a potent dual PI3K/mTOR inhibitor for the treatment of breast cancer. Also, XS-2 was found to be potentially effective against triple-negative breast cancer (TNBC) in vitro during the investigation. We evaluated the in vitro inhibitory effect of XS-2 on 10 cancer cell lines by MTT and 6 kinases to investigated its in vivo antitumor activity in MCF-7 xenograft tumor-bearing BALB/c nude mice. In addition, the in vitro/in vivo toxicity to mice was also assessed by hemolytic toxicity, H&E staining and blood biochemical analysis. In order to investigate the antitumor mechanism of XS-2, a series of experiments were carried out in vitro/in vivo animal model and molecular biological levels such as the cell cycle and the apoptosis assay, real-time PCR, western blot, docking and molecular simulations analysis, etc. What's more, wound healing assay, Transwell and Western Blot were applied to explore the ability of XS-2 to inhibit the cell invasion and migration. The results showed that XS-2 exhibited strong antitumor activity both in vitro and in vivo. The inhibitory activities of XS-2 on ten cancer cell lines were ranging from 1.07 ± 0.11 to 0.002 ± 0.001 µM, which were 1565 times better than that of the lead compound GDC-0941, inhibitory activities against PI3Kα and mTOR kinases were 291.0 and 60.8 nM, respectively. Notably, XS-2 not only showed significant in vivo antitumor activity and low toxicity, with the tumor inhibition rate of 57.0 %, but also exhibited strong inhibitory in the expression of related proteins of PI3K pathway in tumor tissues. In addition, XS-2 significantly inhibited breast cancer MCF-7 and MDA-MB-231 cells in a concentration- and time-dependent manner, and inhibited the migration and invasion ability of MDA-MB-231 and MCF-7 cells. More than that, XS-2 could inhibit the increase of the expression levels of N-cadherin and vimentin upregulated by EGF and reversed the E-cadherin expression down regulated by EGF, resulting in inhibiting EMT in MCF-7 and MDA-MB-231 cells. The results showed that XS-2 was expected to be successfully developed as a high-efficiency and low-toxicity breast cancer therapeutic drug with the potential to inhibit the invasion and migration of TNBC. This provides a new research idea for the treatment of TNBC, which is of great significance.

The role of glycolysis and lactate in the induction of tumor-associated macrophages immunosuppressive phenotype.

Growing evidence highlights that glycolysis and tumor-derived lactate could skew tumor-associated macrophages (TAMs) toward an immunosuppressive phenotype. However, the updated research has not been systematically summarized yet. TAMs are educated by the tumor microenvironment (TME) and exert immunosuppressive functions and tumorigenic effects via multiple biological processes. It is well known that lactate generated by aerobic glycolysis is significantly accumulated in TME and promotes tumor progression in solid tumors. Moreover, some recent research demonstrated that glycolysis is activated in TAMs to support M2-like polarization, which is absolutely in contrast with the metabolic profile of M2 macrophages in inflammation. Notably, lactate produced by high levels of glycolysis is not only a metabolic by-product but also an oncometabolite. TAMs could access the biological information delivered by lactate and further enhance protumor functions such as immunosuppression and angiogenesis. Here, we outline the connection between glycolysis and TAM phenotype to elucidate the metabolic characteristics of TAMs. Further, insights into the specific molecular mechanisms of lactate-induced TAM polarization and potential therapeutic targets are summarized. We sought to discuss the reciprocal interaction between tumor cells and TAMs mediated by lactate, which will lay a foundation for the research aiming to elucidate the complex functions of TAMs.

1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42) inhibits cell proliferation and induces apoptosis via inhibiting ALK and its downstream pathways in Karpas299 cells.

Anaplastic lymphoma kinase (ALK) belongs to the family of receptor tyrosine kinases. Recently, the incidence of anaplastic large cell lymphoma (ALCL) with ALK rearrangement has raised considerably. The application of ALK-targeted inhibitors such as ceritinib provides an effective therapy for the treatment of ALK-positive cancers. However, with the prolongation of treatment time, the emergence of resistance is inevitable. We found that 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42), a novel ceritinib derivative, could inhibit the proliferation of ALK-positive ALCL cells, induce the apoptosis of Karpas299 cells through the mitochondrial pathway in a caspase-dependent manner. In addition, ZX-42 could suppress ALK and downstream pathways including PI3K/Akt, Erk and JAK3/STAT3 and reduce the nuclear translocation of NFκB by inhibiting TRAF2/IKK/IκB pathway. Taken together, our findings indicate that ZX-42 shows more effective activity than ceritinib against ALK-positive ALCL. We hope this study can provide a direction for the structural modification of ceritinib and lay the foundation for the further development of clinical research in ALK-positive ALCL.

A novel tubulin inhibitor, 6h, suppresses tumor-associated angiogenesis and shows potent antitumor activity against non-small cell lung cancers.

Tumor angiogenesis is closely associated with the metastasis and progression of non-small cell lung cancer (NSCLC), a highly vascularized solid tumor. However, novel therapeutics are lacking for the treatment of this cancer. Here, we developed a series of 2-aryl-4-(3,4,5-trimethoxy-benzoyl)-5-substituted-1,2,3-triazol analogs (6a-6x) as tubulin colchicine-binding site inhibitors, aiming to find a novel promising drug candidate for NSCLC treatment. We first identified 2-(2-fluorophenyl)-3-(3,4,5-trimethoxybenzoyl)-5-(3-hydroxyazetidin-1-yl)-2H-1,2,3-triazole (6h) as a hit compound, which inhibited angiogenesis induced by NSCLC cells both in vivo and in vitro. In addition, our data showed that 6h could tightly bind to the colchicine-binding site of tubulin and inhibit tubulin polymerization. We also found that 6h could effectively induce G2/M cell cycle arrest of A549 and H460 cells, inhibit cell proliferation, and induce apoptosis. Furthermore, we showed 6h had the potential to inhibit the migration and invasion of NSCLC cells, two basic characteristics of tumor metastasis. Finally, we found 6h could effectively inhibit tumor progression in A549 xenograft mouse models with minimal toxicity. Taken together, these findings provide strong evidence for the development of 6h as a promising microtubule colchicine-binding site inhibitor for NSCLC treatment.

2-Methoxy-5((3,4,5-trimethosyphenyl) seleninyl) phenol causes G2/M cell cycle arrest and apoptosis in NSCLC cells through mitochondrial apoptotic pathway and MDM2 inhibition.

Nonsmall cell lung cancer (NSCLC) is one of the most common malignancies and needs novel and effective chemotherapy. In this study, our purpose is to explore the anticancer effects of 2-methoxy-5((3,4,5-trimethosyphenyl) seleninyl) phenol (SQ) on human NSCLC (A549 and H460) cells. We found that SQ suppressed the proliferation of NSCLC cells in time- and dose-dependent manners, and blocked the cells at G2/M phase, which was relevant to microtubule depolymerization. Additionally, SQ induced A549 and H460 cell apoptosis by activating the mitochondrial apoptotic pathway. Further, we demonstrated that SQ enhanced the generation of reactive oxygen species (ROS), and pretreatment with N-acetyl- L-cysteine (NAC) attenuated SQ-induced cell apoptosis. Meanwhile, SQ mediated-ROS generation caused DNA damage in A549 and H460 cells. Our data also revealed that SQ-induced apoptosis was correlated with the inhibition of mouse double minute 2 (MDM2) in A549 and H460 cells. In summary, our research indicates that the novel compound SQ has great potential for therapeutic treatment of NSCLC in future.

Design, synthesis and biological evaluation of colchicine glycoconjugates as tubulin polymerization inhibitors.

A series of new colchicine glycoconjugates as tubulin polymerization inhibitors were designed by targeting strategy based on Warburg effect. All of the colchicine glycoconjugates were synthesized and then evaluated for their antiproliferative activities against three human cancer lines HT-29, MCF-7 and Hep-3B. Among them, 1e exhibited greater than 10 times selectivity between GLUT1 highly expressed cells (HT-29 and MCF-7) and GLUT1 lowly expressed cells (Hep-3B), and also showed lower cytotoxicity against HUVECs compared with colchicine. Moreover, 1e significantly inhibited tubulin polymerization and disrupted microtubule networks. GLUT1 inhibitor-dependent cytotoxicity assay demonstrated that the uptake of 1e was regulated via GLUT1. Molecular docking studies showed that 1e could be a substrate of GLUT1 and bind to the colchicine site of tubulin.

The anti-MDR efficacy of YAN against A549/Taxol cells is associated with its inhibition on glycolysis and is further enhanced by 2-deoxy-d-glucose.

Aerobic glycolysis is a hallmark of malignant tumor. Here, the hyperactive glycolysis in multidrug-resistant A549/Taxol cells was demonstrated to be essential for maintaining the vigorous cell viability and drug resistance. 5-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-3-amine (YAN), a newly synthesized tubulin inhibitor, could not only inhibit the glycolysis in A549 and A549/Taxol cells through down-regulating the glycolysis-related proteins, but also disrupt the mitochondrial localization of hexokinase-2 (HK-2) which is related with the apoptosis resistance. The effects of YAN above were relevant to the down-regulation of PI3K-Akt-c-Myc/HIF-1α pathway. Moreover, YAN induced the reactive oxygen species generation in A549 and A549/Taxol cells, which only mediated the apoptosis in A549 cells. We also showed that 2-DG, the glycolysis inhibitor, synergistically enhanced YAN-triggered apoptosis in A549/Taxol cells via further suppressing glycolysis and reducing mitotic slippage. Collectively, we illustrate the inhibition effect of YAN on the glycolysis in A549 and A549/Taxol cells, and provide a fresh insight into the mechanism for the development of YAN as a candidate for multidrug resistant cancer treatment. The finding that 2-DG improved the anti-tumor efficacy of YAN against A549/Taxol cells, offers a reference for solving mitotic slippage-mediated drug resistance.

Novel microtubule inhibitor SQ overcomes multidrug resistance in MCF-7/ADR cells by inhibiting BCRP function and mediating apoptosis.

The occurrence of multidrug resistance (MDR) is one of the impediments in the clinical treatment of breast cancer, and MDR breast cancer has abnormally high breast cancer resistance protein (BCRP/ABCG2) expression. However, there are currently no clinical drugs that inhibit this target. Our previous study found that 2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061/SQ), a small molecule drug with low toxicity to normal tissues, could target microtubules, inhibit the proliferation of breast cancer, and reduce its migration and invasion abilities. However, the effect and the underlying mechanism of SQ on MDR breast cancers are still unknown. Therefore, in this study, we investigated the effect of SQ on adriamycin-resistant MCF-7 (MCF-7/ADR) cells and explored the underlying mechanism. The MTT assay showed that SQ had potent cytotoxicity to MCF-7/ADR cells. In particular, the results of western blot and flow cytometry proved that SQ could effectively inhibit the expression of BCRP in MCF-7/ADR cells to decrease its drug delivery activity. In addition, SQ could block the cell cycle at G2/M phase in parental and MCF-7/ADR cells, thereby mediating cell apoptosis, which was related with the inhibition of PI3K-Akt-MDM2 pathway. Taken together, our findings indicate that SQ overcomes multidrug resistance in MCF-7/ADR cells by inhibiting BCRP function and mediating apoptosis through PI3K-Akt-MDM2 pathway inhibition.

Targeted inhibition of acidic nucleoplasmic DNA-binding protein 1 enhances radiosensitivity of non-small cell lung cancer.

Acidic nucleoplasmic DNA binding protein 1 (AND-1, also known as WD repeat and HMG-box DNA-binding protein 1, WDHD1) plays an important role in DNA replication and repair, but the relationship between AND-1 and radiosensitivity is not well understood. This research explored the impact of AND-1 on the radiosensitivity of non-small cell lung cancer (NSCLC) for the first time. NSCLC cells were treated with AND-1 siRNA or a new AND-1 inhibitor, CH-3, and clonogenic survival assay was used to characterize cell radiosensitivity. Cell cycle and apoptosis were examined by flow cytometry. DNA damage was detected by comet assay, immunofluorescence, and homologous recombination (HR) repair assay. Finally, the radiosensitization effect of CH-3 was investigated in vivo in a xenograft tumor model. The results showed that AND-1 inhibition significantly increased the radiosensitivity of NSCLC cells. Mechanistically, AND-1 inhibitor (CH-3) induced G2/M phase arrest by regulating the ATM signaling pathway and enhanced irradiation-induced DNA damage by inhibiting the DNA HR repair pathway. CH-3 enhanced the radiosensitivity of NSCLC cells in vivo. The development of radiosensitizers that target AND-1 may provide an alternative strategy to inhibit NSCLC.