PTPRT loss enhances anti-PD-1 therapy efficacy by regulation of STING pathway in non-small cell lung cancer.

With the revolutionary progress of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer, identifying patients with cancer who would benefit from ICIs has become critical and urgent. Here, we report protein tyrosine phosphatase receptor type T (PTPRT) loss as a precise and convenient predictive marker independent of PD-L1 expression for anti-PD-1/PD-L1 axis therapy. Anti-PD-1/PD-L1 axis treatment markedly increased progression-free survival in patients with PTPRT-deficient tumors. PTPRT-deficient tumors displayed cumulative DNA damage, increased cytosolic DNA release, and higher tumor mutation burden. Moreover, the tyrosine residue 240 of STING was identified as a direct substrate of PTPRT. PTPRT loss elevated phosphorylation of STING at Y240 and thus inhibited its proteasome-mediated degradation. PTPRT-deficient tumors released more IFN-ß, CCL5, and CXCL10 by activation of STING pathway and increased immune cell infiltration, especially of CD8 T cells and natural killer cells, ultimately enhancing the efficacy of anti-PD-1 therapy in multiple subcutaneous and orthotopic tumor mouse models. The response of PTPRT-deficient tumors to anti-PD-1 therapy depends on the tumor-intrinsic STING pathway. In summary, our findings reveal the mechanism of how PTPRT-deficient tumors become sensitive to anti-PD-1 therapy and highlight the biological function of PTPRT in innate immunity. Considering the prevalence of PTPRT mutations and negative expression, this study has great value for patient stratification and clinical decision-making.

Erratum: ßKlotho is identified as a target for theranostics in non-small cell lung cancer: Erratum.

[This corrects the article DOI: 10.7150/thno.35582.].

Durable response to low-dose pralsetinib in a renal insufficient patient with NSCLC harboring concurrent CCDC6-RET, LINCO1264-RET, and SEMA5A-RET fusions: A case report.

INTRODUCTION: RET-rearranged fusions have been considered as oncogenic drivers in 1% to 2% of non-small cell lung cancers (NSCLC). ARROW study has demonstrated a new selective RET tyrosine kinase inhibitors (TKIs) shows remarkable and durable responses in RET-rearranged NCSLC. In this study mainly recruited patients with common fusion partners KIF5B and CCDC6. There is still a lack of definitive conclusions about effective of rare RET fusion variants to anti-RET therapies. CASE REPORT: A Chinese 58-year-old female renal insufficient patient with no history of smoking was diagnosed as stage IIIA (T2N2M0) lung adenocarcinoma. Next-generation sequencing targeting 520 cancer-related genes was performed on the pleural effusion samples and revealed 2 novel RET fusions LINCO1264-RET and SEMA5A-RET, concomitant with a common CCDC6-RET. MANAGEMENT AND OUTCOME: The patient was first treated with multiple lines of chemotherapy and switched to lenvatinib but failed to respond. Due to renal insufficiency, she subsequently received pralsetinib with gradually reduced dosages (400 mg-300 mg-200 mg-100 mg qd) and achieved a partial response (PR) lasting for more than 10 months, accompanied by the declined allele frequencies of all 3 RET fusions. DISCUSSION/CONCLUSIONS: We reported the first case of the pralsetinib efficacy in NSCLC with 3 concurrent RET fusions. Our case also indicates the sensitivity of the newly identified RET fusions to this RET selective inhibitor pralsetinib, and highlights the low-dose treatment option for patients with renal insufficient background.

SMO mutation predicts the effect of immune checkpoint inhibitor: From NSCLC to multiple cancers.

Background: The emergence of immune checkpoint inhibitors (ICIs) is one of the most promising breakthroughs for the treatment of multiple cancer types, but responses vary. Growing evidence points to a link between developmental signaling pathway-related genes and antitumor immunity, but the association between the genomic alterations in these genes and the response to ICIs still needs to be elucidated. Methods: Clinical data and sequencing data from published studies and our cohort were collected to analyze the association of the mutation status of SMO with the efficacy of ICI therapy in the non-small cell lung cancer (NSCLC) cohort and the pan-cancer cohort. Furthermore, the correlation between SMO mutation and immunotherapeutic biomarkers such as immune cell infiltration, immune-related genes, and underlying signaling pathways was analyzed. Three SMO mutant plasmids were transfected into cells to explore the SMO mutation status in the context of its expression and cell growth. Result: In the NSCLC discovery cohort, the median progression-free survival in the SMO mutant (SMO_MUT) was longer than that in the wild type (SMO_WT) (23.0 vs. 3.8 months, adjusted p = 0.041). This finding was further confirmed in the NSCLC validation cohort (8.7 vs. 5.1 months, adjusted p = 0.013). In the pan-cancer cohort (n = 1,347), a significant overall survival advantage was observed in patients with SMO mutations [not reached (NR) vs. 18 months, adjusted p = 0.024]. In the subgroup analysis, the survival advantage of SMO_MUT against SMO_WT was prominent and consistent across genders, ages, treatment types, cancer types, and the tumor mutation burden (TMB) status (all p interaction > 0.05). In an in vitro experiment, we found that both the mutant and wild-type plasmids can promote the expression of SMO, but the mutant plasmid had lower SMO mRNA and protein levels than the wild type. In CCK-8 experiments, we found that SMO_MUT plasmids can improve the growth of Calu-1 and PC-9 cells, but this capability varied between different mutations and cells. Upon further exploration, the SMO mutation status was found to be related to a higher TMB, more neoantigen load, more DNA damage repair (DDR) mutations, higher microsatellite instability (MSI) score, and higher CD8+ T-cell infiltration. Conclusions: The SMO mutation status is an independent prognostic factor that can be used to predict better clinical outcomes of ICI treatment across multiple cancer types.

Comprehensive analysis of MET mutations in NSCLC patients in a real-world setting.

Background: Aberrant mesenchymal-epithelial transition/hepatocyte growth factor (MET/HGF) regulation presented in a wide variety of human cancers. MET exon 14 skipping, copy number gain (CNG), and kinase domain mutations/arrangements were associated with increased MET activity, and considered to be oncogenic drivers of non-small cell lung cancers (NSCLCs). Methods: We retrospectively analyzed 564 patients with MET alterations. MET alterations were classified into structural mutations or small mutations. MET CNG, exon 14 skipping, gain of function (GOF) mutations, and kinase domain rearrangement were defined as actionable mutations. Results: Six hundred thirty-two MET mutations were identified including 199 CNG, 117 exon 14 skipping, 12 GOF mutations, and 2 actionable fusions. Higher percentage of MET structural alterations (CNG + fusion) were detected in advanced NSCLC patients. Moreover, MET CNG was enriched while exon 14 skipping was rare in epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI)-treated advanced NSCLC patients. Ten of the 12 MET GOF mutations were also in EGFR-TKI-treated patients. Fifteen (68.1%) of the 22 patients treated with crizotinib or savolitinib had a partial response. Interestingly, one patient had a great response to savolitinib with a novel MET exon 14 skipping mutation identified after failure of immune-checkpoint inhibitor. Conclusions: Half of the MET alterations were actionable mutations. MET CNG, exon 14 skipping and GOF mutations had different distribution in different clinical scenario but all defined a molecular subgroup of NSCLCs for which MET inhibition was active.

FGFR1 promotes tumor immune evasion via YAP-mediated PD-L1 expression upregulation in lung squamous cell carcinoma.

BACKGROUND: Variations in FGFR1 are common driver mutations of LSQCC. And immune checkpoint inhibitors targeting PD-1 and PD-L1 are powerful anticancer weapons. Activation of FGFR1 leads to tumorigenesis through multiple downstream molecules, including YAP, but whether and how FGFR1 regulates tumor immune evasion remain largely unclear. METHODS: LSQCC cells were modified to increase or decrease the expression of FGFR1, YAP and PD-L1, as assessed by molecular assays. After FGFR1 knockdown, cancer cells were assessed after cocultured with Jurkat T cells in vitro, and the tumor microenvironment were analyzed in C57BL/6 mice. The effect of the combination of FGFR1 knockdown and PD-1 blockade was also explored. RESULTS: In human LSQCC, activation of FGFR1 was positively correlated with transcription of PD-L1. In H520 and HCC95 cells, FGFR1 upregulated PD-L1 expression via YAP, and YAP initiated the transcription of PD-L1 after binding to its promoter region. FGFR1 knockdown decreased tumor growth, reduced immune escape and induced reactivation of CD8+ T cells. The combination of FGFR1 knockdown and PD-1 blockade synergistically exerted antitumor effects. CONCLUSIONS: The FGFR1/YAP/PD-L1 regulatory axis mediates tumor-associated immune suppression in lung squamous cell carcinoma, and FGFR1 knockdown reactivates T cells in the tumor microenvironment. Synergistic inhibition of both FGFR1 and PD-1/PD-L1 pathways may be a possible treatment for lung cancer patients.

Homologous recombination deficiency (HRD) can predict the therapeutic outcomes of immuno-neoadjuvant therapy in NSCLC patients.

BACKGROUND: Neoadjuvant immunotherapy is emerging as novel effective intervention in lung cancer, but study to unearth effective surrogates indicating its therapeutic outcomes is limited. We investigated the genetic changes between non-small cell lung cancer (NSCLC) patients with varied response to neoadjuvant immunotherapy and discovered highly potential biomarkers with indicative capability in predicting outcomes. METHODS: In this study, 3 adenocarcinoma and 11 squamous cell carcinoma NSCLC patients were treated by neoadjuvant immunotherapy with variated regimens followed by surgical resection. Treatment-naive FFPE or fresh tissues and blood samples were subjected to whole-exome sequencing (WES). Genetic alternations were compared between differently-responded patients. Findings were further validated in multiple public cohorts. RESULTS: DNA damage repair (DDR)-related InDel signatures and DDR-related gene mutations were enriched in better-responded patients, i.e., major pathological response (MPR) group. Besides, MPR patients exhibited provoked genome instability and unique homologous recombination deficiency (HRD) events. By further inspecting alternation status of homology-dependent recombination (HR) pathway genes, the clonal alternations were exclusively enriched in MPR group. Additionally, associations between HR gene alternations, percentage of viable tumor cells and HRD event were identified, which orchestrated tumor mutational burden (TMB), mutational intratumor heterogeneity (ITH), somatic copy number alteration (SCNA) ITH and clonal neoantigen load in patients. Validations in public cohorts further supported the generality of our findings. CONCLUSIONS: We reported for the first time the association between HRD event and enhanced neoadjuvant immunotherapy response in lung cancer. The power of HRD event in patient therapeutic stratification persisted in multifaceted public cohorts. We propose that HR pathway gene status could serve as novel and additional indicators guiding immune-neoadjuvant and immunotherapy treatment decisions for NSCLC patients.

Treatment preferences for epidermal growth factor receptor mutation-positive non-small cell lung cancer with brain metastasis: a large-scale survey from Chinese oncologists.

Background: Radiotherapy combined with tyrosine kinase inhibitor (TKI) has drawn extensive attention as a treatment regimen for patients with epithelial growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC) with brain metastases (BMs). However, the optimal regimens and treatment sequence remain unknown. This study sought to investigate the opinions of Chinese oncologists toward the regimen selection and therapeutic timing for patients with EGFR-mutated NSCLC with BMs. Methods: A survey was developed by the expert group of the Specialty Committee of Lung Cancer of the Chinese Anti-Cancer Association. Between January and March 2018, the survey was distributed in online and paper forms to oncologists working in departments that may receive patients with NSCLC with BMs. Results: The survey was completed by 1,000 oncologists. When selecting a patient's therapeutic regimen, respondents were most likely to consider the benefit to overall survival (32%), followed by the benefit to progression-free survival (18%) and quality of life (17%). Radiotherapy combined with EGFR-TKI agents is the leading regimen over monotherapy (46-58%), with rates increasing in patients with neurological symptoms and a higher number of intracranial metastases. For patients with 1-3 BMs, stereotactic radiosurgery (SRS) with TKI was the preferred regimen. For patients with >3 BMs, whole-brain radiotherapy with TKI was the preferred regimen in accordance with the preference towards meningeal BM. Conclusions: Radiotherapy combined with EGFR-TKI agents is the preferred regimen among Chinese oncologists for the treatment of patients with EGFR mutation-positive NSCLC with BMs. BM number and type may influence the selection of radiotherapy regimen. Randomized controlled trials could be helpful in addressing current disputes regarding treatment regimens.

NLRP4 negatively regulates type I interferon response and influences the outcome in anti-programmed cell death protein (PD)-1/PD-ligand 1 therapy.

The challenge to improve the clinical efficacy and enlarge the population that benefits from immune checkpoint inhibitors (ICIs) for non-small-cell lung cancer (NSCLC) is significant. Based on whole-exosome sequencing analysis of biopsies from NSCLC patients before anti-programmed cell death protein-2 (PD-1) treatment, we identified NLRP4 mutations in the responders with a longer progression-free survival (PFS). Knockdown of NLRP4 in mouse Lewis lung cancer cell line enhanced interferon (IFN)-α/ß production through the cGAS-STING-IRF3/IRF7 axis and promoted the accumulation of intratumoral CD8+ T cells, leading to tumor growth retardation in vivo and a synergistic effect with anti-PD-ligand 1 therapy. This was consistent with clinical observations that more tumor-infiltrating CD8+ T cells and elevated peripheral IFN-α before receiving nivolumab treatment were associated with a longer PFS in NSCLC patients. Our study highlights the roles of tumor-intrinsic NLRP4 in remodeling the immune contextures in the tumor microenvironment, making regional type I IFN beneficial for ICI treatment.

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma.

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3+, CD4+ and CD8+ T cells in spleens and lower PD-1 expression in CD4+ and CD8+ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.

Standardization of pleural effusion-based tumor mutation burden (TMB) estimation using capture-based targeted sequencing.

BACKGROUND: Tumor mutation burden (TMB) has received considerable attention as a potential predictive biomarker for response to anticancer treatment with immune checkpoint inhibitors (ICIs), and has been increasingly incorporated into clinical practice. Currently, TMB is often determined with tissue biopsies using whole-exome sequencing (WES) or panel-based targeted sequencing. Meanwhile, liquid biopsies such as blood are actively investigated as alternative media, although there is currently no report of the performance of targeted sequencing in assessing TMB using pleural effusion (PE) specimens. METHODS: Thirty-two patients diagnosed with advanced non-small cell lung cancer (NSCLC) with associated PE were prospectively enrolled (NCT03546452). Cell-free DNA (cfDNA) from the supernatant of PE was subjected to both WES and capture-based targeted sequencing using various commercially-available panels. RESULTS: All five panels assessed in this study demonstrated a good correlation with WES-derived TMB, with correlation coefficients ranging from 0.68-0.81. Two- and three-tier classification systems built on the TMB estimates achieved respective concordance rates of 74% and 63% between classifications based on WES- and panel-derived TMB levels. CONCLUSIONS: This study provides real-world evidence that all panels assessed in this study can be used for TMB evaluation based on PE samples. We also demonstrated that PE can serve as an alternative medium for TMB evaluation. To the best of our knowledge, this is the first study evaluating the potential of PE samples for TMB estimation, thereby providing a basis for establishing future standard protocols.

Distinct profile of cell-free DNA in malignant pleural effusion of non-small cell lung cancer and its impact on clinical genetic testing.

Cell-free DNA (cfDNA) in supernatant of pleural effusion from advanced NSCLC patients has been proved as surrogate sample detecting therapeutic targets as well as tumor mutation burden (TMB). As recently reported, cfDNA in pleural effusion supernatant is superior to plasma in TMB evaluation. It is reasonable to hypothesize that cfDNA profile in pleural effusion (PE) and plasma might be different. It remains to be elucidated why cfDNA in PE supernatant impacts on genetic analysis. Consequently, the approach dealing with cfDNA from PE supernatant might need to be different from that for plasma cfDNA in order to obtain accurate clinical genetic testing result. Methods: Pleural effusion samples from 32 patients with stage IV lung adenocarcinoma were collected. Supernatant and sediment were processed separately to extract Cell-free DNA as well as sediment DNA (PE-S). cfDNA from pleural effusion was analyzed by Agilent 2100 bioanalyzer. Libraries were prepared by 1) direct use of the total cfDNA without fragmentation step (PE-FL) or 2) use of full-length cfDNA fragmented to 150-250bp (PE-F), 3) use of cfDNA fragments enriched to ~167bp (PE-E167) as well as 4) use of cfDNA fragments larger than 500bp enriched (PE-E500). All samples were subjected to targeted next-generation sequencing (NGS) with a panel of 448 cancer-related genes as well as a panel of 10 NSCLC driver genes. Results: cfDNA were successfully extracted from 30 MPE samples. cfDNA displayed distinct profile in supernatant of malignant pleural effusion from that of plasma cfDNA. No statistical difference in detection of hotspot variations between PE-E167 and PE-F by 448-gene or 10-gene panel. While TMB from PE-F samples was significantly higher than that from PE-E167 and PE-FL. Higher TMB from PE-F was resulted from cancer-unspecific variants with low allele frequency (0.1%-1%) which were mainly introduced by long-fragment cfDNA. Similar genetic profile was observed between paired cfDNA of PE-FL and cfDNA of PE-E167. Conclusion: Long-fragment cfDNA in the PE supernatant will introduce low abundant cancer unrelated variants which leads overestimation of TMB. Paired PE-FL and PE-E167 gave comparable outcomes. Direct use of the total cfDNA without fragmentation step (PE-FL) is recommended for library preparation of NGS testing in clinical practice to exclude interference from long fragments of the cfDNA.

Correction: FGFR1-ERK1/2-SOX2 axis promotes cell proliferation, epithelial-mesenchymal transition, and metastasis in FGFR1-amplified lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PAK5 promotes the cell stemness ability by phosphorylating SOX2 in lung squamous cell carcinomas.

Growing evidences suggest that the overexpression of p21-activated kinase 5 (PAK5) plays an important role in various tumor progression. However, the role of PAK5 and its downstream target gene(s) in lung squamous cell carcinomas (LUSC) are waiting to be elucidated. TCGA data were utilized to evaluate the expression levels of PAK5 in LUSC. We then explored the role of PAK5 in maintaining the stem-like phenotype of lung squamous cancer cells through RT-PCR, flow cytometry, oncosphere-forming assay. In addition, co-immunoprecipitation, western blotting and immunofluorescence assays were used to determine SOX2 as a novel effector of PAK5. Xenograft models in nude mice were established to explore the roles of PAK5 in lung cancer growth. In this study, we have shown that PAK5 is overexpressed in LUSC tissues. The absence of PAK5 abolishes self-renewal ability of LUSC cells by decreasing the expression and phosphorylation of SOX2 in vitro and in vivo. In xenograft models, knockdown or pharmacological inhibition of PAK5 suppressed the tumor growth and metastasis of lung squamous cancer cells in vivo. Taken together, our findings suggest that the PAK5-mediated SOX2 phosphorylation promoted the cancer stem cell-like phenotype of LUSC cells. PAK5 inhibition may be a promising target in the treatment of SOX2 positive lung squamous cell cancer.

Erratum: ßKlotho is identified as a target for theranostics in non-small cell lung cancer: Erratum.

[This corrects the article DOI: 10.7150/thno.35582.].

Enhanced autocrine FGF19/FGFR4 signaling drives the progression of lung squamous cell carcinoma, which responds to mTOR inhibitor AZD2104.

Lung cancer occurrence and associated mortality ranks top in all countries. Despite the rapid development of targeted and immune therapies, many patients experience relapse within a few years. It is urgent to uncover the mechanisms that drive lung cancer progression and identify novel molecular targets. Our group has previously identified FGF19 as a prognostic marker and potential driver gene of lung squamous cell carcinomas (LSQ) in Chinese smoking patients. However, the underlying mechanism of how FGF19 promotes the progression of LSQ remains unclear. In this study, we characterized and confirmed that FGF19 serves as an oncogenic driver in LSQ development and progression, and reported that the amplification and high expression of FGF19 in LSQ was significantly associated with poor overall and progression-free survival. A higher serum level of FGF19 was found in lung cancer patients, which could also serve as a novel diagnostic index to screen lung cancer. Overproduction of FGF19 in LSQ cells markedly promoted cell growth, progression and metastasis, while downregulating FGF19 effectively inhibited LSQ progression in vitro and in vivo. Moreover, downregulating the receptor FGFR4 was also effective to suppress the growth and migration of LSQ cells. Since FGF19 could be induced by smoking or endoplasmic reticulum stress, to tackle the more malignant FGF19-overproducing LSQ, we reported for the first time that inhibiting mTOR pathway by using AZD2014 was effective and feasible. These findings have offered a new strategy by using anti-FGF19/FGFR4 therapy or mTOR-based therapy in FGF19-driven LSQ.

ßKlotho is identified as a target for theranostics in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains a great challenge, calling for the identification of novel molecular targets with diagnostic/therapeutic value. Here, we sought to characterize the expression of ßKlotho and its anti-tumor roles in NSCLC. Methods: The expression of ßKlotho was examined in NSCLC cells and tissues by western blot, qRT-PCR and immunohistochemistry staining respectively. Biological roles of ßKlotho were revealed by a series of functional in vitro and in vivo studies. Serum ßKlotho concentrations of patients were measured using specific ELISA methods. Results: Serum ßKlotho concentrations of NSCLC patients were significantly lower than the control group. Moreover, ßKlotho expression was negatively associated with lymph node metastasis, overall survival and progression-free survival. Overexpression of ßKlotho or exogenous ßKlotho administration inhibited the proliferation and migration of NSCLC cells, accompanied by induction of apoptosis, G1 to S phase arrest, and inactivation of ERK1/2, AKT and STAT3 signaling. Furthermore, ßKlotho overexpression inhibited NSCLC tumor growth in vivo. Conclusions: ßKlotho serves as a novel target for theranostics in NSCLC, which has potential clinical applications in the future.

Reciprocal regulatory mechanism between miR-214-3p and FGFR1 in FGFR1-amplified lung cancer.

MicroRNA (miRNA) and fibroblast growth factor receptor 1 (FGFR1) dysregulation are considered to play an important role in tumor proliferation, invasion, and metastasis. However, the regulatory mechanism between miRNAs and FGFR1 in lung cancer remains unclear and extremely critical. miR-214-3p was sharply decreased and showed a significantly negative correlation with FGFR1 in lung cancer patients (n = 30). Luciferase reporter assay confirmed that miR-214-3p could downregulate FGFR1 by directly targeting 3'-untranslated region (UTR). miR-214-3p inhibited the processes of epithelial-mesenchymal transition and Wnt/MAPK/AKT (Wnt/mitogen-activated protein kinase/AKT) signaling pathway by targeting FGFR1. Moreover, miR-214-3p not only established a negative feedback regulation loop with FGFR1 through ERK (extracellular signal-regulated kinase) but also developed a synergism with FGFR1 inhibitor AZD4547. In conclusion, our study demonstrated the regulatory mechanism between miR-214-3p and FGFR1 in lung cancer. miR-214-3p acts as a vital target in FGFR1-amplified lung cancer by forming a miR-214-3p-FGFR1-Wnt/MAPK/AKT signaling pathway network. Co-targeting miR-214-3p and FGFR1 could provide greater benefits to patients with FGFR1-amplified lung cancer.

Exosomal miR-499a-5p promotes cell proliferation, migration and EMT via mTOR signaling pathway in lung adenocarcinoma.

Tumor-derived exosomes contain informative microRNAs involved in carcinogenesis, cell migration, invasion and epithelial-mesenchymal transition (EMT), eventually contributing to metastasis of cancers. This study aims to clarify which and how exosomal miRNA affects tumor carcinogenesis and metastasis. Among them, miR-499a-5p was upregulated in both highly metastatic lung cancer cell line and their exosomes. MiR-499a-5p overexpression promoted cell proliferation, migration and EMT, while miR-499a-5p knockdown suppressed these processes in vitro. Inhibition of miR-499a-5p by antagomirs administration restrained tumor growth in vivo. Consequently, miR499a-sufficient exosomes, derived from highly metastatic cell line, enhanced cell proliferation, migration and EMT via mTOR pathway, and the effect could be inhibited by miR-499a-5p inhibitor. The study reveals the potential diagnostic and therapeutic value of cancer-derived exosomal miR-499a-5p, and sheds a new insight on a novel molecular mechanism which modulates metastasis.

FGFR1-ERK1/2-SOX2 axis promotes cell proliferation, epithelial-mesenchymal transition, and metastasis in FGFR1-amplified lung cancer.

Epithelial-mesenchymal transition (EMT) is an important process for cancer metastasis, drug resistance, and cancer stem cells. Activation of fibroblast growth factor receptor 1 (FGFR1) was found to promote EMT and metastasis in prostate and breast cancers, but the effects and mechanisms in lung cancer was unclear. In this study, we aimed to explore whether and how activation of FGFR1 promotes EMT and metastasis in FGFR1-amplified lung cancer. We show that activation of FGFR1 by its ligand fibroblast growth factor 2 (FGF2) promoted proliferation, EMT, migration, and invasion in FGFR1-amplified lung cancer cell lines H1581 and DMS114, whereas inhibition of FGFR1 suppressed these processes. FGFR1 activation upregulated expression of Sry-related HMG box 2 (SOX2) by downstream phosphorylated ERK1/2; moreover, the upregulation of SOX2 by autophosphorylation variant ERK2_R67S plasmid transfection was not suppressed by FGFR1 inhibitor AZD4547 or MEK/ERK inhibitor AZD6244 in vitro. And SOX2 expression was also significantly upregulated in ERK2_R67S lentivirus-transfected stable cell lines in vivo. Overexpression of SOX2 promoted cell proliferation, EMT, migration, and invasion. Importantly, activation of FGFR1 could not promote these processes in SOX2-silenced stable cell lines. In orthotopic and subcutaneous lung cancer xenograft models, inhibition of FGFR1 suppressed tumor growth, SOX2 expression, EMT, and metastasis in vivo; however, these processes caused by SOX2-overexpressing stable cell lines were not suppressed by FGFR1 inhibition. Higher expression of FGFR1 and SOX2 were positively correlated, and both were associated with shorter survival in lung cancer patients. In conclusion, our findings reveal that activation of FGFR1 promotes cell proliferation, EMT, and metastasis by the newly defined FGFR1-ERK1/2-SOX2 axis in FGFR1-amplified lung cancer.