M2-like macrophage-derived exosomes facilitate metastasis in non-small-cell lung cancer by delivering integrin αVß3.

Metastasis is the most prevalent cause of cancer deaths, and immunological components of the tumor microenvironment, especially tumor-associated macrophages (TAMs), play a vital role in cancer metastasis. However, the underlying mechanisms of TAMs on non-small-cell lung cancer (NSCLC) metastasis remain largely unexplored. Herein, we demonstrated that M2-like TAMs facilitate the migration and invasion of cancer cells in vitro and in vivo through intercellular delivery of M2-like macrophage-derived exosomes (M2-exos). Importantly, we found that M2-exos had considerably higher levels of integrin (ITG) αV and ß3. The impact of M2-like macrophage-mediated invasion and migration of NSCLC cells was clearly decreased when ITG αVß3 was blocked. Mechanistically, exosomal ITG αVß3 produced from M2-like macrophages successfully triggered the focal adhesion kinase signaling pathway in recipient cells, boosting the migratory and invasive abilities of NSCLC cells. Clinically, we found that metastatic NSCLC patients had greater ITG αV and ß3 expression, which was associated with a worse prognosis. This study reveals a novel mechanism by which M2-exos significantly increased NSCLC cell migration and invasion by delivering integrin αVß3. Exosomal ITG αVß3 can be used as a potential prognostic marker, and blocking ITG αVß3 could be a viable treatment option for preventing tumor metastasis.

Exosomes of A549 Cells Induced Migration, Invasion, and EMT of BEAS-2B Cells Related to let-7c-5p and miR-181b-5p.

As carriers containing abundant biological information, exosomes could deliver the property of donor cells to recipient cells. Emerging studies have shown that tumor cells could secrete a mass of exosomes into the microenvironment to regulate bystander cells. However, the underlying mechanisms of such a phenomenon remain largely unexplored. In this research, we purified and identified the exosomes of A549 cells and found that A549-cell-derived exosomes promoted BEAS-2B cells migration, invasion, and epithelial-mesenchymal transition (EMT). Importantly, we observed that let-7c-5p and miR-181b-5p were attenuated in A549-cell-derived exosomes compared to BEAS-2B-cell-derived exosomes. The analysis of miRNA expression level in BEAS-2B cells indicated that incubation with A549-cell-derived exosomes reduced the expression levels of let-7c-5p and miR-181b-5p. In transient transfections assay, we found that downregulation of let-7c-5p and miR-181b-5p simultaneously showed stronger promotion of BEAS-2B cells migration and invasion than individually. Moreover, exosomes secreted from A549 cells with upregulated expression of let-7c-5p and miR-181b-5p significantly reduce their regulatory effect on BEAS-2B cells. Bioinformatics analyses revealed that let-7c-5p and miR-181b-5p inhibit the EMT process mainly by regulating focal adhesion and mitogen-activated protein kinase (MAPK) signaling pathway. Thus, our data demonstrated that A549-cell-derived exosomal let-7c-5p and miR-181b-5p could induce migration, invasion, and EMT in BEAS-2B cells, which might be regulated through focal adhesion and MAPK signaling pathway. The expression level of let-7c-5p and miR-181b-5p may show great significance for the early diagnosis of lung cancer.

PD0325901, an ERK inhibitor, enhances the efficacy of PD-1 inhibitor in non-small cell lung carcinoma.

ERK pathway regulated the programmed death ligand-1 (PD-L1) expression which was linked to the response of programmed death-1 (PD-1)/PD-L1 blockade therapy. So it is deducible that ERK inhibitor could enhance the efficacy of PD-1 inhibitor in cancer immunotherapy. In this study, PD0325901, an oral potent ERK inhibitor, strongly enhanced the efficacy of PD-1 antibody in vitro and in vivo models in non-small cell lung carcinoma (NSCLC) cells. Mechanistically, PD0325901 or shRNA-ERK1/2 significantly downregulated the PD-L1 expression in NSCLC cells and increased the CD3+ T cells infiltration and functions in tumor tissue. There was a positive correlation between the p-ERK1/2 expression and PD-L1 expression in patients with NSCLC. And the patients with low p-ERK1/2 expression were observed a high response rate of PD-1/PD-L1 blockage therapy. Our results demonstrate that PD0325901, an ERK inhibitor, can enhance the efficacy of PD-1 blockage against NSCLC in vitro and in vivo models. And the combination of ERK inhibitor such as PD0325901 and PD-1/PD-L1 blockage is a promising regimen and encouraged to be further confirmed in the treatment of patients with NSCLC.

The key roles of cancer stem cell-derived extracellular vesicles.

Cancer stem cells (CSCs), the subpopulation of cancer cells, have the capability of proliferation, self-renewal, and differentiation. The presence of CSCs is a key factor leading to tumor progression and metastasis. Extracellular vesicles (EVs) are nano-sized particles released by different kinds of cells and have the capacity to deliver certain cargoes, such as nucleic acids, proteins, and lipids, which have been recognized as a vital mediator in cell-to-cell communication. Recently, more and more studies have reported that EVs shed by CSCs make a significant contribution to tumor progression. CSCs-derived EVs are involved in tumor resistance, metastasis, angiogenesis, as well as the maintenance of stemness phenotype and tumor immunosuppression microenvironment. Here, we summarized the molecular mechanism by which CSCs-derived EVs in tumor progression. We believed that the fully understanding of the roles of CSCs-derived EVs in tumor development will definitely provide new ideas for CSCs-based therapeutic strategies.

Intercellular transfer of exosomal wild type EGFR triggers osimertinib resistance in non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR)-mutated lung cancer constitutes a major subgroup of non-small cell lung cancer (NSCLC) and osimertinib is administrated as first-line treatment. However, most patients with osimertinib treatment eventually relapse within one year. The underlying mechanisms of osimertinib resistance remain largely unexplored. METHODS: Exosomes isolation was performed by differential centrifugation. Co-culture assays were conducted to explore the alteration of drug sensitivity by cell viability and apoptosis assays. Immunofluorescence and flow cytometry were performed to visualize the formation or absorption of exosomes. Exosomes secretion was measured by Nanoparticle Tracking Analysis or ELISA. The xenograft tumor model in mice was established to evaluate the effect of exosomes on osimertinib sensitivity in vivo. RESULTS: Intercellular transfer of exosomal wild type EGFR protein confers osimertinib resistance to EGFR-mutated sensitive cancer cells in vitro and in vivo. Co-culture of EGFR-mutated sensitive cells and EGFR-nonmutated resistant cells promoted osimertinib resistance phenotype in EGFR-mutated cancer cells, while depletion of exosomes from conditioned medium or blockade of exosomal EGFR by neutralizing antibody alleviated this phenotype. Mechanistically, osimertinib promoted the release of exosomes by upregulated a Rab GTPase (RAB17). Knockdown of RAB17 resulted in the decrease of exosomes secretion. Moreover, exosomes could be internalized by EGFR-mutated cancer cells via Clathrin-dependent endocytosis and then the encapsulated exosomal wild type EGFR protein activated downstream PI3K/AKT and MAPK signaling pathways and triggered osimertinib resistance. CONCLUSIONS: Intercellular transfer of exosomal wild type EGFR promotes osimertinib resistance in NSCLC, which may represent a novel resistant mechanism of osimertinib and provide a proof of concept for targeting exosomes to prevent and reverse the osimertinib resistance.

Pristimerin induces apoptosis and inhibits proliferation, migration in H1299 Lung Cancer Cells.

Background: The natural occurring pristimerin, a quinonemethide triterpenoid, is extracted from a variety of species of the Celastraceae and Hippocrateaceae family. This research investigated the in vitro anti-cancer potential of pristimerin on NSCLC cells NCI-H1299 and elucidated the molecular mechanism. Methods: Cell growth inhibition by pristimerin was assessed using the MTT assay. Apoptosis was detected using the Annexin V/propidium iodide (PI) test. The colony forming assay was used to investigate the anti-proliferative effects of pristimerin. Wound healing assay and the transwell cell migration assay were utilized to determine the inhibitory effects of migration and invasion, respectively. Western blot was used to detect the protein expression, and real-time-quantitative (RT-q) PCR was used to analyze the mRNA expression. Results: The results showed that pristimerin inhibited the proliferation of H1299 cells with an IC50 value of 2.2 ± 0.34 µM and induced apoptosis in a dose-dependent manner. The colony formation ability was reduced in a dose-dependent manner. A marked inhibition of migration and invasion against H1299 cells was observed in a dose- or time-dependent manner. Moreover, the decreased protein levels of vimentin, F-actin, integrin ß1, matrix metalloproteinase (MMP2) and Snail revealed the potential inhibition of epithelial-to-mesenchymal transition (EMT). The regulated mRNA levels of integrin ß1, MMP2 and Snail indicated the great potential in the treatment of NSCLC. Conclusion: In conclusion, our study demonstrated that pristimerin suppressed NSCLC cells NCI-H1299 in vitro, exhibited potent activities of proliferation inhibition and apoptosis induction. Furthermore, the treatment of pristimerin decreased migration and invasion of H1299, which was correlated with EMT-related proteins and mRNA.

Adverse Effects of Anti-PD-1/PD-L1 Therapy in Non-small Cell Lung Cancer.

Currently, immunotherapy has shown great efficacy in clinical trials, and monoclonal antibodies directed against immune checkpoint PD-1/PD-L1 have shown encouraging results in first-line or second-line treatment of non-small cell lung cancer patients. Meanwhile, anti-PD-1/PD-L1 immune checkpoint drugs combined with other treatments, such as chemotherapy, targeted therapy as well as anti-CTLA-4 checkpoint therapy, are considered an attractive treatment with higher efficacy. However, toxicity associated with PD-1/PD-L1 blockade is worth attention. Understanding the adverse effects caused by anti-PD-1/PD-L1 immunosuppressive agents is vital to guide the clinical rational use of drug. In this review, we summarized the adverse effects that occurred during the clinical use of anti-PD-1/PD-L1 inhibitors in the treatment of non-small cell lung cancer and discussed how to effectively manage and respond to these adverse reactions.

The Biological Functions and Clinical Applications of Integrins in Cancers.

Integrins are the adhesion molecules and receptors of extracellular matrix (ECM). They mediate the interactions between cells-cells and cells-ECM. The crosstalk between cancer cells and their microenvironment triggers a variety of critical signaling cues and promotes the malignant phenotype of cancer. As a type of transmembrane protein, integrin-mediated cell adhesion is essential in regulating various biological functions of cancer cells. Recent evidence has shown that integrins present on tumor cells or tumor-associated stromal cells are involved in ECM remodeling, and as mechanotransducers sensing changes in the biophysical properties of the ECM, which contribute to cancer metastasis, stemness and drug resistance. In this review, we outline the mechanism of integrin-mediated effects on biological changes of cancers and highlight the current status of clinical treatments by targeting integrins.

Exosomes with low miR-34c-3p expression promote invasion and migration of non-small cell lung cancer by upregulating integrin α2ß1.

Exosomes play critical roles in regulating various physiological and pathological processes, including immune stimulation, immune suppression, cardiovascular diseases, and cancers. Recent studies show that exosomes that transport specific microRNAs (miRNAs) are involved in tumor development. However, the molecular mechanism by which tumor invasion and migration are regulated by exosomes from non-small cell lung cancer (NSCLC) is not well understood. Here, we show that exosomes shuttling low levels of miR-34c-3p are involved in NSCLC progression. Our results showed that exosomes derived from NSCLC cells carrying low levels of miR-34c-3p could be transported into the cytoplasm of NSCLC cells and accelerate NSCLC invasion and migration by upregulating integrin α2ß1. A luciferase assay revealed that integrin α2ß1 was the direct target of miR-34c-3p, and overexpression of integrin α2ß1 could promote the invasion and migration of NSCLC cells. The analysis of exosomes derived from clinical serum samples indicated that the expression of miR-34c-3p was significantly downregulated in exosomes from NSCLC patients compared with that of normal controls. A549-derived exosomes promoted NSCLC cells lung metastases in vivo. Exosomes shuttling low levels of miR-34c-3p were associated with the progression of NSCLC in vitro and in vivo. Our data demonstrate that exosomes shuttling low levels of miR-34c-3p can accelerate the invasion and migration of NSCLC by upregulating integrin α2ß1. MiR-34c-3p can be a diagnostic and prognostic marker for NSCLC. High expression of integrin α2ß1 is positively related to the migration and metastasis of NSCLC cells.

The Role of Exosomal microRNA in Cancer Drug Resistance.

Exosomes affect the initiation and progression of cancers. In the tumor microenvironment, not only cancer cells, but also fibroblasts and immunocytes secrete exosomes. Exosomes act as a communicator between cells by transferring different cargos and microRNAs (miRNAs). Drug resistance is one of the critical factors affecting therapeutic effect in the course of cancer treatment. The currently known mechanisms of drug resistance include drug efflux, alterations in drug metabolism, DNA damage repair, alterations of energy programming, cancer stem cells and epigenetic changes. Many studies have shown that miRNA carried by exosomes is closely associated with the development of drug resistance mediated by the above-mentioned mechanisms. This review article will discuss how exosomal miRNAs regulate the drug resistance.

Costunolide induces apoptosis and inhibits migration and invasion in H1299 lung cancer cells.

Costunolide being a sesquiterpene lactone, is known to have anticancer properties. The present study investigated the anticancer effects of costunolide against the H1299 human non­small­cell lung cancer (NSCLC) cell line. Inhibition of cell viability by costunolide was assessed via a MTT assay. Furthermore, the apoptotic rate was detected using Annexin V/propidium iodide labeling. A colony forming cell assay was performed to investigate the antiproliferative effects of costunolide. Wound healing and Transwell assays were performed to determine the inhibitory effects of costunolide on migration and invasion, respectively. Western blot analysis was undertaken to determine protein expression, and reverse transcription­quantitative PCR was performed to assess mRNA expression levels. The results demonstrated that costunolide inhibited the viability of H1299 cells, with a half maximal inhibitory concentration value of 23.93±1.67 µM and induced cellular apoptosis in a dose­dependent manner. Furthermore, the colony formation, migrative and invasive abilities of the H1299 cells were inhibited in a dose­ or time­dependent manner. The protein expression levels of E­cadherin increased and those of N­cadherin decreased following treatment with costunolide, which suggested that costunolide inhibited epithelial­to­mesenchymal transition. The mRNA levels of B­Raf, E­cadherin, N­cadherin, integrins α2 and ß1, as well as matrix metalloproteinases 2 were also found to be regulated costunolide. These findings indicate the potential of costunolide in the treatment of NSCLC.

Anti-Cancer Effects of Pristimerin and the Mechanisms: A Critical Review.

As a quinonemethide triterpenoid extracted from species of the Celastraceae and Hippocrateaceae, pristimerin has been shown potent anti-cancer effects. Specifically, it was found that pristimerin can affect many tumor-related processes, such as apoptosis, autophagy, migration and invasion, vasculogenesis, and drug resistance. Various molecular targets or signaling pathways are also involved, such as cyclins, reactive oxygen species (ROS), microRNA, nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways. In this review, we will focus on the research about pristimerin-induced anti-cancer activities to achieve a deeper understanding of the targets and mechanisms, which offer evidences suggesting that pristimerin can be a potent anti-cancer drug.

The targeting of non­coding RNAs by curcumin: Facts and hopes for cancer therapy (Review).

Curcumin [(1E,6E)­1,7­bis(4­hydroxy­3­-methoxyphenyl) hepta­1,6­diene­3,5­dione] is a natural polyphenol that is derived from the turmeric plant (curcuma longa L.). Curcumin is widely used in food coloring, preservatives, and condiments. Curcumin possesses anti­tumor, anti­oxidative and anti­inflammatory efficacy, as well as other pharmacological effects. Emerging evidence indicates that curcumin alters microRNAs (miRNAs) and long non­coding RNAs (lncRNAs) in various types of cancers. Both miRNAs and lncRNAs are non­coding RNAs that can epigenetically modulate the expression of multiple genes via post­transcriptional regulation. In the present review, the interactions between curcumin and non­coding RNAs are summarized in numerous types of cancers, including lung, colorectal, prostate, breast, nasopharyngeal, pancreatic, blood, and ovarian cancer, and the vital non­coding RNAs and their downstream targets are described.