The formation of an immunosuppressive microenvironment and up-regulation of PD-L1 protein are the main causes of tumor immune escape. Previous reports suggest that Angiotensin II (Ang II) can modulate the immune status of tumor microenvironment in non-small cell lung cancer (NSCLC), but the underlying mechanism remains not fully understood. Here we demonstrated that AngII treatment causes the reduction of intratumoral infiltrating CD4 T lymphocytes in tumor-bearing mice, increases the accumulation of immunosuppressive granulocytes and TAMs in tumor tissue, and upregulates the expression levels of immunosuppressive marker genes. In addition, AngII/AGTR1 axis triggers cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability by human antigen R (HuR), an AU-rich element (ARE)-binding protein. Collectively, AngII/AGTR1 signaling promotes the tumor immunosuppressive microenvironment by upregulating PD-L1 in NSCLC, the mechanism of which is largely accounted by HuR-mediated PD-L1 mRNA stabilization.

Aim: To determine the role of claudin-3 in cancer stemness in nonsquamous non-small-cell lung carcinoma (NSCLC). Materials & methods: In vitro/vivo extreme limiting dilution analysis and the side population assay were used to investigate the role of claudin-3 in regulating cancer stemness in nonsquamous NSCLC. Results & conclusion: Claudin-3 depletion decreased the formation rates of spheres and tumors and increased cisplatin sensitivity. Claudin-3 was also identified as one downstream target of estrogen receptor-α in regulating cancer stemness. Moreover, targeting CLDN-3 transcription by small molecules including withaferin A, estradiol and fulvestrant suppressed cancer stemness and reversed chemoresistance. These results demonstrated claudin-3 is one positive regulator of cancer stemness in nonsuqamous NSCLC.

Background and Purpose In non-small-cell lung carcinoma (NSCLC) patients, the L858R/T790M mutation of the epithelial growth factor receptor (EGFR) is a major cause of acquired resistance to EGFR-TKIs treatment that limits their therapeutic efficacy. Identification of drugs that can preferentially kill the NSCLC harbouring L858R/T790M mutation is therefore critical. Here, we have evaluated the effects of ursolic acid, an active component isolated from herbal sources, on erlotinib-resistant H1975 cells that harbour the L858R/T790M mutation. Experimental Approach Gene expression omnibus (GEO) profiles analyses was applied to detect differentially expressed genes in NSCLC cells harbouring EGFR mutation. AnnexinV-FITC/PI, TUNEL staining, MTT, wound healing, RT-PCR, qRT-PCR, western blots, immunostaining, dual-luciferase reporters and ChIP-PCR were utilized to investigate the effects of ursolic acid in vitro and in vivo. Key Results The cancer/testis antigen family 45 member A2 (CT45A2) was highly expressed in H1975 cells. Ectopic expression of CT45A2 in H1975 cells increased cell proliferation and motility in vitro. Silencing the CT45A2 expression strongly attenuated H1975 cells motility and growth. The anti-cancer effect of ursolic acid was critically dependent on CT45A2 expression in H1975 cells. Ursolic acid suppressed CT45A2 gene transcription mediated by transcriptional factor TCF4 and beta-catenin signalling. Conclusions and Implications CT45A2 is a novel oncogene for NSCLC with an EGFR T790 mutation. Ursolic acid induced apoptosis and inhibited proliferation of H1975 cells by negatively regulating the beta-catenin/TCF4/CT45A2 signalling pathway. Therefore, ursolic acid may be a potential candidate treatment for NSCLC harbouring the EGFR-L858R/T790M mutation.

Adriamycin (ADM) is a commonly used drug in clinical breast cancer treatment. However, some breast cancer types or breast cancers subjected to repeated ADM exposure develop strong resistance to ADM thus limiting its clinical efficacy. In this study, we found for the first time that toosendanin (TSN), a triterpenoid extracted from the traditional Chinese medicine Melia toosendan Sieb et Zucc, could successfully reverse adriamycin resistance in human breast cancer cells. Immunofluorescence and HPLC analysis demonstrated that TSN promoted adriamycin accumulation in breast cancer cells, especially in the nucleus. Furthermore, TSN could significantly reduce ABCB1 expression. We then found that TSN was capable of suppressing adriamycin-induced Akt phosphorylation, probably due to downregulation of the PI3K catalytic subunits P110α and P110β, and inhibition of DNA-PKcs. Importantly, the inhibitory effect of TSN on PI3K P110α and P110β expression was specifically observed in breast cancer cells but not in normal human cells. Moreover, TSN significantly potentiated the anti-cancer effect of ADM in the 4T1 breast cancer model and its inhibition rate was nearly 90%. Thus, TSN could be used as a novel PI3K inhibitor to reverse breast cancer resistance. The combination of ADM and TSN may represent a useful strategy for human breast cancer therapy.

Musashi-1 (Msi1) is an evolutionarily conserved RNA-binding protein that has been reported to be the key regulator in malignancies and with involvement in cancer stemness. In the present study, a novel Msi1 transcript variant generated by alternative splicing was identified and termed Msi1 variant 2. This variant was observed to be ubiquitously expressed in cancerous and non-cancerous cells compared with its wild-type variant, which is preferentially expressed in cancer cells. Notably, the expression levels of Msi1 variant 2 were inversely associated with the protein expression levels of Msi1 in various cancer cells. This naturally truncated variant contains 899 nucleotides and a skipping event of exons 3 and 4, which leads to the emergence of a premature TGA stop codon in exon 5. The present results also demonstrated that hypoxia increased the resistance of H460 cells to cisplatin by suppressing the exon 3 and 4 skipping event of Msi1. In summary, the present study identified a novel splice variant of Msi1 lacking two complete RNA recognition motifs, and revealed the role of exon 3 and 4 skipping of Msi1 pre-mRNA in regulating cisplatin resistance under hypoxia. These observations indicate that targeting Msi1 alternative splicing could represent a valuable strategy to repress Msi1 signaling in tumors overexpressing this RNA-binding protein.

Cancer stem-like cells (CSCs) have been reported to play major roles in tumorigenesis, tumor relapse, and metastasis after therapy against colorectal carcinoma (CRC). Therefore, identification of colorectal CSC regulators could provide promising targets for CRC. Ligand-of-Numb protein X1 (LNX1) is one E3 ubiquitin ligase which mediates the ubiquitination and degradation of Numb. Although several studies indicate LNX1 could be a potential suppressor of cancer diseases, the functions of LNX1 in mediating cancer stemness remain poorly understood. In this study, LNX1 was identified as a negative regulator of cancer stemness in CRC, which was downregulated in colonospheres or side population (SP) cells. Furthermore, the coxsackievirus and adenovirus receptor (CXADR) was found to be one critical downstream mediator of cancer stemness regulated by LNX1. Interestingly, the anti-breast cancer drug tamoxifen was found to be an agonist of LNX1 and suppress cancer stemness in CRC. In sum, this study provided the evidences that LNX1 signaling plays important roles in regulating the stemness of colon cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively triggers cancer cell death via its association with death receptors on the cell membrane, but exerts negligible side effects on normal cells. However, some non-small-cell lung carcinoma (NSCLC) patients exhibited resistance to TRAIL treatment in clinical trials, and the mechanism varies. In this study, we described for the first time that toosendanin (TSN), a triterpenoid derivative used in Chinese medicine for pain management, could significantly sensitize human primary NSCLC cells or NSCLC cell lines to TRAIL-mediated apoptosis both in vitro and in vivo, while showing low toxicity against human primary cells or tissues. The underlying apoptotic mechanisms involved upregulation of death receptor 5 (DR5) and CCAAT/enhancer binding protein homologous protein, which is related to the endoplasmic reticulum stress response, and is further associated with reactive oxygen species generation and Ca2+ accumulation. Surprisingly, TSN also induced autophagy in NSCLC cells, which recruited membrane DR5, and subsequently antagonized the apoptosis-sensitizing effect of TSN. Taken together, TSN can be used to sensitize tumors and the combination of TRAIL and TSN may represent a useful strategy for NSCLC therapy; moreover, autophagy serves as an important drug resistance mechanism for TSN.

In this study, a shikonin ester derivative, compound , was selected to evaluate its anticancer activities and we found that compound exhibited better antitubulin activities against the human HepG2 cell line with an IC50 value of 1.097 μM. Furthermore, the inhibition of tubulin polymerization results indicated that compound demonstrated the most potent antitubulin activity (IC50  = 13.88), which was compared with shikonin and colchicine as positive controls (IC50  = 25.28 μM and 22.56 μM), respectively. Compound was simulated to have good binding site with tubulin and arrested the cell cycle at G2/M phase, which also induces apoptosis in HepG2 cells, in which P53 and members of Bcl-2 protein family were both involved in the progress of apoptosis revealed by western blot. Confocal microscopy observations revealed compound targeted tubulin and altered its polymerization by interfering with microtubule organization. Based on these results, compound functions as a potent anticancer agent targeting tubulin.

General control nonderepressible kinase 2 (GCN2) is a promising target for cancer therapy. However, the role of GCN2 in cancer cell survival or death is elusive; further, small molecules targeting GCN2 signaling are not available. By using a GCN2 level-based drug screening assay, we found that GCN2 protein level critically determined the sensitivity of the cancer cells toward Na + ,K + -ATPase ligand–induced apoptosis both in vitro and in vivo, and this effect was largely dependent on C/EBP homologous protein (CHOP) induction. Further analysis revealed that GCN2 is a short-lived protein. In A549 lung carcinoma cells, cellular β-arrestin1/2 associated with GCN2 and maintained the GCN2 protein level at a low level by recruiting the E3 ligase NEDD4L and facilitating consequent proteasomal degradation. However, Na + ,K + -ATPase ligand treatment triggered the phosphorylation of GCN2 at threonine 899, which increased the GCN2 protein level by disrupting the formation of GCN2–β-arrestin–NEDD4L ternary complex. The enhanced GCN2 level, in turn, aggravated Na + ,K + -ATPase ligand–induced cancer cell apoptosis. Our findings reveal that GCN2 can exert its proapoptotic function in cancer cell death by posttranslational mechanisms. Moreover, Na + ,K + -ATPase ligands emerge as the first identified small-molecule drugs that can trigger cancer cell death by modulating GCN2 signaling.

Increasing evidence demonstrated that Na + ,K + -ATPase ligands, also called as cardiotonic steroids, are hormone-like immunoregulators, because endogenous Na + ,K + -ATPase ligands are frequently detected in inflammatory-related diseases, moreover, Na + ,K + -ATPase ligands regulate multiple aspects of immune responses. One of prominent roles of Na + ,K + -ATPase ligands in regulating immunity is their abilities of modulating cytokines expression. Na + ,K + -ATPase ligands can either upregulate or downregulate IL-1β, TNF-α, IL6, or iNOS expressions in different model system, however, all of those studies pointed to transcriptional upregulation. In our recent studies, we proposed for the first time that Na + ,K + -ATPase ligands are capable of regulating cytokines mRNA stability by integrating multiple posttranscriptional mechanisms, including human antigen R (HuR) translocation, generation of miR181s, and formation of stress granules. These mechanisms did not function alone, but act in a synergistic or an antagonistic manner to fine-tune the cytokines expression, HuR nuclear export, however, forms signalosome and plays a core role among these processes. By taking advantage of these posttranscriptional mechanisms, Na + ,K + -ATPase ligands stabilized cyclooxygenase-2 mRNA stability in lung epithelial cells and induced acute lung injury. In monocytes, ouabain-induced HuR export competed with miR181s on the shared target of TNF-α, also triggered stress granules formation and recruited TNF-α mRNA into it for protection, thereby stabilizing TNF-α mRNA and reversing sepsis-induced immunoparalysis, both in vitro and in vivo. Besides in immune-related diseases, HuR also regulated a variety of pro-oncogenes and anti-oncogenes expressions in cancer cells, which determined the cancer cells sensitivity towards Na + ,K + -ATPase ligands or other chemotherapeutic drugs. In sum, HuR emerges as a very important signaling molecule in Na + ,K + -ATPase ligands-mediated effects, which opens new avenues for understanding of the pathophysiologic and pharmacological activities of Na + ,K + -ATPase ligands. Identification of the components of HuR signalosome will offer more novel targets and biomarkers for diseases therapy.

Shikonin has previously been reported to function as a potent anti-cancer drug that induces cell apoptosis via diverse pathways. To optimize the effectiveness of its pro-apoptotic functions, shikonin was chosen as the best compound for obtaining shikonin ester derivatives (3a–3n) because it possesses an ester group on the side chain hydroxyl position, and the biological activity was evaluated as a potent anti-proliferating inhibitor for many cancer cell lines. Among these compounds, compound 3j exhibited better anti-cancer activities against human hepatocellular carcinoma cell line (HepG2) with an IC50 value of 0.759 μM which was better than that of shikonin which was 1.288 μM. The flow cytometry results showed that compound 3j inhibited the cell growth and caused the cell cycle to be arrested at the G2/M phase. Meanwhile, obvious apoptosis induced by 3j was observed using the Annexin V combined with propidium iodide assay, showing that 3j induced apoptosis of HepG2 cells in a dose and time dependent manner. To investigate the underlying mechanism in the process of apoptosis induced by 3j, the western blot technique was used and the cleavage of caspase-9 was observed but not that of caspase-8. Furthermore, the elevated expression of the p53 protein was positively correlated with the decreased Bcl-2 protein levels and increased Bax protein and the cytochrome C levels. This indicated that the mitochondrial apoptosis pathway directed by p53 responded in the process of apoptosis was induced by 3j. Based on these data, we conclude that compound 3j has the best anti-proliferating and pro-apoptotic effect among the 14 newly synthesized compounds, and it could be a potent candidate for cancer therapy.

Immunoparalysis is an important pathological mechanism in sepsis. However, an effective small molecule therapy is lacking. Here, we show that ouabain, a Na(+),K(+)-ATPase ligand, can reverse immunoparalysis in vitro, in vivo, and in clinical samples. Notably, the effect of ouabain was critically dependent on TNF-α expression. However, ouabain had opposing effects on the stability of TNF-α mRNA: Ouabain triggered miR-181 transcription, which promoted TNF-α mRNA degradation and induced immunoparalysis, and ouabain triggered the nuclear export of human antigen R (HuR), which stabilized TNF-α mRNA and suppressed immuno-paralysis. Interestingly, because the miR-181 binding site is located within the HuR binding site in the 3'-untranslated region of TNF-α, in ouabain-treated cells, HuR competed with miR-181 for binding to TNF-α mRNA and recruited TNF-α mRNA to stress granules, thereby stabilizing TNF-α mRNA and reversing immunoparalysis. Ouabain also induced GM-CSF and interferon-γ expression in a HuR-dependent manner. Hence, the fine-tuning of TNF-α mRNA stability by HuR and miR181 plays a crucial role in immunoparalysis, and Na(+),K(+)-ATPase ligands are promising agents for immunoparalysis therapy.