Different treatment efficacies and T790M acquisition of EGFR-TKIs on NSCLC patients with variable Del-19 subtypes of EGFR.

EGFR exon 19 deletion (Del-19) comprises multiple advanced NSCLC subtypes. EGFR-tyrosine kinase inhibitor (TKI) efficacy and T790M acquisition in various Del-19 subtypes is unknown. We prospectively collected tissue samples from patients harboring NSCLC with Del-19 between 2006 and 2020. We evaluated EGFR-TKI treatment effectiveness among the different Del-19 subtypes. We collected 1391 NSCLC samples from 892 patients with Del-19, and the most common subtype was del E746-A750 (67.5%). 741 patients had taken first- or second-generation EGFR-TKIs. There were no significant differences in response rates between patients with different Del-19 subtypes (P = .630). Patients with indel E746 had the longest median PFS (14.6 months), but those with non-LRE deletions had the shortest PFS (8.9 months; P = .002). For OS analysis, patients with indel E746 also had the longest OS (34.1 months), but those with non-LRE deletions had the shortest OS (21.1 months; P = .046). Patients with different Del-19 subtypes showed no significant differences in the T790M acquisition rates (P = .443). Among the 151 patients with acquired T790M who received third-generation EGFR-TKIs, the Del-19 subtype was not associated with different RR and PFS. In vitro cellular viability and activation of the EGFR pathway analysis were consistent with the clinical findings. In conclusion, compared with del E746-A750, indel E746 was associated with longer PFS and OS, but the non-LRE subtype was correlated with shorter survival prognosis. There were no significant differences in the acquired T790M rate and treatment effectiveness of subsequent third-generation EGFR-TKIs between various Del-19 subgroups.

LncRNA SLCO4A1-AS1 suppresses lung cancer progression by sequestering the TOX4-NTSR1 signaling axis.

BACKGROUND: Metastasis is a multistep process involving the migration and invasion of cancer cells and is a hallmark of cancer malignancy. Long non-coding RNAs (lncRNAs) play critical roles in the regulation of metastasis. This study aims to elucidate the role of the lncRNA solute carrier organic anion transporter family member 4A1-antisense 1 (SLCO4A1-AS1) in metastasis and its underlying regulatory mechanisms. METHODS: A comprehensive analysis of the Gene Expression Omnibus (GEO) database were used to identify metastasis-associated lncRNAs. Transwell migration and invasion assays, and a tail vein-injection mouse model were used to assess the migration and invasion of cancer cells in vitro and in vivo, respectively. High-throughput screening methods, including MASS Spectrometry and RNA sequencing (RNA-seq), were used to identify the downstream targets of SLCO4A1-AS1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blotting, RNA pull-down, RNA immunoprecipitation (RIP), fluorescence in situ hybridization (FISH), and chromatin immunoprecipitation (ChIp) assays were conducted to identify and validate the underlying regulatory mechanisms of SLCO4A1-AS1. RESULTS: SLCO4A1-AS1 reduced cancer cell migration and invasion by disrupting cytoskeleton filaments, and was associated with longer overall survival in patients with lung adenocarcinoma. SLCO4A1-AS1 directly interacted with the DNA-binding protein, TOX High Mobility Group Box Family Member 4 (TOX4), to inhibit TOX4-induced migration and invasion. Furthermore, RNA-seq revealed that neurotensin receptor 1 (NTSR1) is a novel and convergent downstream target of SLCO4A1-AS1 and TOX4. Mechanistically, SLCO4A1-AS1 functions as a decoy of TOX4 by interrupting its interaction with the NTSR1 promoter and preventing NTSR1 transcription. Functionally, NTSR1 promotes cancer cell migration and invasion through cytoskeletal remodeling, and knockdown of NTSR1 significantly inhibits TOX4-induced migration and invasion. CONCLUSION: These findings demonstrated that SLCO4A1-AS1 antagonizes TOX4/NTSR1 signaling, underscoring its pivotal role in lung cancer cell migration and invasion. These findings hold promise for the development of novel therapeutic strategies targeting the SLCO4A1-AS1/TOX4/NTSR1 axis as a potential avenue for effective therapeutic intervention in lung cancer.

Prognostic value of early and late spontaneous conversion into a shockable rhythm for patients with out-of-hospital cardiac arrest.

BACKGROUND: The prognostic significance of conversion into a shockable rhythm in patients who experienced out-of-hospital cardiac arrest (OHCA) with an initially nonshockable rhythm is controversial, perhaps due to the timing of rhythm conversion not being considered previously. We aimed to compare the different prognoses of patients with OHCA and early and late conversion of their rhythm into a shockable rhythm. METHODS: This was a single-centre retrospective cohort study. We enrolled patients with OHCA who were sent to a medical centre in central Taiwan from 2016 to 2020. Patients <18 years old, those with cardiac arrest due to trauma or a circumstantial cause, and those for whom resuscitation was not attempted were excluded. Patients were divided into two groups in accordance with presentation with an initially shockable rhythm. Those with an initially nonshockable rhythm were divided into three subgroups: early-conversion, late-conversion, and nonconversion groups. The primary outcome was the neurological functional status upon discharge from hospital. RESULTS: A total of 1645 patients with OHCA were included: initially shockable rhythm group, 339; early conversion group, 68; late-conversion group, 166; and nonconversion group, 1072. After adjustment, multivariate logistic regression revealed that a favourable neurological outcome was more common in the early conversion group than the nonconversion group (odds ratio [OR] 2.4; 95% confidence interval [CI], 1.1-5.3; p = 0.035), whereas the late-conversion group did not significantly differ from the nonconversion group (OR 0.5; 95% CI, 0.1-1.5; p = 0.211). The proportions of sustained return of spontaneous circulation and survival to discharge were also higher in the early conversion group than the late-conversion group (OR 2.9 95% CI 1.6-5.5, p = 0.001 and OR 4.5, 1.8-11.0, p = 0.001, respectively). CONCLUSION: In patients who experience OHCA and have an initially nonshockable rhythm, early conversion into a shockable rhythm resulted in a better prognosis, whereas late conversion was not significantly different from nonconversion.

Acquired resistance to EGFR tyrosine kinase inhibitors is mediated by the reactivation of STC2/JUN/AXL signaling in lung cancer.

Constitutive activation of the epidermal growth factor receptor (EGFR) signaling pathway is implicated in the initiation and progression of lung cancer. EGFR tyrosine kinase inhibitor (TKI)-targeted therapy has become the standard treatment for nonsmall cell lung cancer (NSCLC) patients. However, acquired resistance to these agents remains a major obstacle for managing NSCLC. Here, we investigated a novel strategy to overcome EGFR TKI resistance by targeting the stanniocalcin 2 (STC2)-JUN-AXL pathway. We revealed that STC2 was expressed at significantly higher levels in EGFR TKI-resistant cells. Further, clinical analysis showed that STC2 expression was increased after the development of EGFR TKI resistance and that higher levels were correlated with shorter progression-free survival in EGFR TKI-treated lung cancer patients. Moreover, STC2 overexpression in EGFR TKI-sensitive cells resulted in EGFR TKI resistance. Conversely, genetic silencing of STC2 rendered EGFR TKI-resistant cells more sensitive to EGFR TKIs. Mechanically, STC2 enhanced AXL promoter activity by increasing the phosphorylation of c-Jun, which is an indispensable transcription factor that transactivates AXL. STC2 promoted activation of the JUN-AXL-extracellular signal-regulated kinase (ERK) signaling axis in lung cancer cells. Pharmacological or genetic inhibition of AXL-ERK activity inhibited STC2-mediated EGFR TKI resistance. We also demonstrated that PE2988 cells, a C797S-independent osimertinib-resistant primary cancer cell line from a lung cancer patient, responded to combined AXL inhibitor and osimertinib treatment. In conclusion, our research indicates that STC2 overexpression is important for acquired resistance to EGFR TKIs and that STC2-JUN-AXL-ERK signaling might be a potential therapeutic target to overcome resistance to EGFR TKIs.

Heparin co-factor II enhances cell motility and promotes metastasis in non-small cell lung cancer.

Using the Serial Analysis of Gene Expression (SAGE) database from the Cancer Genome Anatomy Project, we identified heparin co-factor II (HCII), which is over-expressed in non-small cell lung cancer (NSCLC). Here, we investigated the clinical significance of HCII and provided molecular evidence to support the suggestion that HCII could enhance cancer metastasis in NSCLC. We found that high HCII expression in tumour tissue was associated with increased cancer recurrence and shorter overall survival times in 75 clinically operable NSCLC patients. High pretreatment plasma concentration of HCII was associated with reduced overall survival in 57 consecutive NSCLC patients. We over-expressed and knocked down HCII expression in lung cancer cell lines and confirmed that HCII could promote cell motility, invasion ability and filopodium dynamics in NSCLC cells in vitro and increased metastatic colonization in an in vivo mouse model. Exogenous treatment of HCII promoted cancer cell migration, and this promigratory effect of HCII was independent of thrombin. We further showed that HCII could up-regulate cancer cell migration through the activation of PI3K, which acts upstream of Rac1 and Cdc42, and this effect could be blocked by heparin. We suggest that HCII is a novel metastasis enhancer and may be used as a prognostic predictor for heparin treatment in NSCLC.

Urinary transforming growth factor α and serum α-fetoprotein as tumor markers of hepatocellular carcinoma.

This case-control study aimed to evaluate the diagnostic application of urinary transforming growth factor (TGF) α and serum α-fetoprotein (AFP) in hepatocellular carcinoma (HCC). TGFα and AFP were determined in 90 pairs of age- and gender-matched patients with cirrhotic HCC and patients with cirrhosis alone and 60 healthy controls. The results indicated that TGFα and AFP levels in patients with HCC were higher than in those with cirrhosis alone or healthy controls (each P = 0.0001). Multivariate analysis indicated that TGFα (odds ratio (OR) 1.03, 95% confidence interval (CI) 1.05-1.16) and AFP (OR 1.03, 95% CI 1.01-1.06) were closely associated, in a dose-related fashion, with the development of HCC. The optimal cutoff values, determined with the receiver operating characteristic (ROC) curves, were 29 µg/g creatinine for TGFα and 100 ng/ml for AFP, respectively. The areas under ROC curve (AUC) were 0.74 for TGFα and 0.78 for AFP, respectively. Both biomarkers showed the same sensitivity (52.2%), high specificity, high positive predictive value, and moderate positive likelihood ratio. Determination of both markers in parallel significantly increased the AUC (0.91) and diagnostic accuracy (92.2%), with a high sensitivity (86.7 %), specificity (97.8%), positive predictive value (PPV; 97.5%), and moderate positive likelihood ratio (PLR; 39.4). Among 31 cirrhotic HCC with AFP ≤ 20 ng/ml, the calculated AUC for TGFα was 0.79, with a sensitivity of 64.5%, specificity of 96.7%, PPV of 87.0%, and PLR of 19.5. In conclusion, urinary TGFα and serum AFP are complementary tumor markers for detection of HCC with low AFP production.

CD44s and CD44v8-10 isoforms confer acquired resistance to osimertinib by activating the ErbB3/STAT3 signaling pathway.

AIMS: Although epidermal growth factor receptor (EGFR)-mutant lung cancers respond well to osimertinib, acquired resistance to osimertinib eventually develops through EGFR-dependent and EGFR-independent resistance mechanisms. CD44 splicing variants are widely expressed in lung cancer tissues. However, it remains unclear whether specific splicing variants are involved in acquired resistance to osimertinib. MAIN METHODS: The real-time PCR was performed to measure the expression levels of total CD44 and specific CD44 splicing variants (CD44s or CD44v). Gene knockdown and restoration were performed to investigate the effects of CD44 splicing variants on osimertinib sensitivity. Activation of the signaling pathway was evaluated using receptor-tyrosine-kinase phosphorylation membrane arrays, co-immunoprecipitation, and western blotting. KEY FINDINGS: Clinical analysis demonstrated that the expression level of total CD44 increased in primary cancer cells from lung adenocarcinomas patients after the development of acquired resistance to osimertinib. Furthermore, osimertinib-resistant cells showed elevated levels of either the CD44s variant or CD44v variants. Manipulations of CD44s or CD44v8-10 were performed to investigate their effects on treatment sensitivity to osimertinib. Knockdown of CD44 increased osimertinib-induced cell death in osimertinib-resistant cells. However, restoration of CD44s or CD44v8-10 in CD44-knockdown H1975/AZD-sgCD44 cells induced osimertinib resistance. Mechanically, we showed that ErbB3 interacted with CD44 and was transactivated by CD44, that consequently triggered activation of the ErbB3/STAT3 signaling pathway and led to CD44s- or CD44v8-10-mediated osimertinib resistance. SIGNIFICANCE: CD44 is a co-receptor for ErbB3 and triggers activation of the ErbB3 signaling axis, leading to acquired resistance to osimertinib. CD44/ErbB3 signaling may represent a therapeutic target for overcoming osimertinib resistance.

miR-204 suppresses cancer stemness and enhances osimertinib sensitivity in non-small cell lung cancer by targeting CD44.

Osimertinib is an effective treatment option for patients with advanced non-small cell lung cancer (NSCLC) with EGFR activation or T790M resistance mutations; however, acquired resistance to osimertinib can still develop. This study explored novel miRNA-mRNA regulatory mechanisms that contribute to osimertinib resistance in lung cancer. We found that miR-204 expression in osimertinib-resistant lung cancer cells was markedly reduced compared to that in osimertinib-sensitive parental cells. miR-204 expression levels in cancer cells isolated from treatment-naive pleural effusions were significantly higher than those in cells with acquired resistance to osimertinib. miR-204 enhanced the sensitivity of lung cancer cells to osimertinib and suppressed spheroid formation, migration, and invasion of lung cancer cells. Increased miR-204 expression in osimertinib-resistant cells reversed resistance to osimertinib and enhanced osimertinib-induced apoptosis by upregulating BIM expression levels and activating caspases. Restoration of CD44 (the direct downstream target gene of miR-204) expression reversed the effects of miR-204 on osimertinib sensitivity, recovered cancer stem cell and mesenchymal markers, and suppressed E-cadherin expression. The study demonstrates that miR-204 reduced cancer stemness and epithelial-to-mesenchymal transition, thus overcoming osimertinib resistance in lung cancer by inhibiting the CD44 signaling pathway.

Survival of lung adenocarcinoma patients with malignant pleural effusion.

In the era of targeted therapy, the association between lung adenocarcinoma patient survival and malignant pleural effusions (MPEs) remains unclear. This study investigated the clinical characteristics, survival and epidermal growth factor receptor (EGFR) gene (EGFR) mutation status of lung adenocarcinoma patients with MPE. From June 2005 to December 2010, consecutive pleural effusions were collected prospectively. Patient clinical characteristics, EGFR mutation status, and overall survival were analysed. We collected MPEs from 448 patients in stage IV lung adenocarcinoma at initial diagnosis. Median overall survival for patients with MPEs at initial diagnosis and following disease progression were 14.3 months and 21.4 months, respectively (p=0.001). There were 296 (66.1%) patients harbouring EGFR mutations, the mutation rates among patients with an MPE at initial diagnosis and one following disease progression were 68.2% and 56.6%, respectively (p=0.044); the L858R mutation rate was also higher among the former (32.6% versus 18.1%; p=0.009). Multivariate analysis revealed that patients who: developed MPEs following disease progression, harboured EGFR mutations, and received EGFR-tyrosine kinase inhibitor therapy, had longer overall survival. Patients in stage IV lung adenocarcinoma with MPEs at initial diagnosis have shorter overall survival and higher EGFR mutation rate, especially for L858R, than patients who develop MPEs following disease progression.

Α-synuclein and ß-synuclein enhance secretion protein production in baculovirus expression vector system.

The baculovirus expression vector system (BEVS) is widely used as a tool for expressing of recombinant proteins in insect cells or larvae. However, the expression level of secretion pathway proteins is often lower than that of cytosolic and nucleus proteins. Thus, we attempted to improve production of secreted proteins by using a secretory alkaline phosphatase-EGFP fusion protein (SEFP)-based bi-cistronic baculovirus vector to identify chaperones that have potential on boosting secreted protein production. As co-expressed SEFP with a chaperone, calreticulin (CALR), it was found that the secreted SEFP enzyme activity can be boosted up to twofold. This result demonstrated the SEFP-based bi-cistronic approach can be used to identify the genes that can enhance secretion protein production in BEVS. Thus, the chaperone activity of α-synuclein (α-syn) and ß-synuclein (ß-syn) was evaluated in cells co-expressed with SEFP and compared that with CALR by analyzing localization, alkaline phosphatase enzyme activity, and mRNA expression levels of SEFP. Our results showed that SEFP enzyme activity from cells co-expressed with both synuclein proteins can be enhanced up to 2.3-fold and this increment was better than that caused by CALR. Moreover, this enhancement might arise from the transcription enhancement or higher RNA stability. By this novel approach, we provided evidences that α- and ß-syn can enhance secretion proteins production in BEVS.

MiR-503 pleiotropically regulates epithelial-mesenchymal transition and targets PTK7 to control lung cancer metastasis.

OBJECTIVE: In lung cancer patients, most deaths are caused by the distant dissemination of cancer cells. Epithelial-mesenchymal transition (EMT) and collective cell migration are distinct and important mechanisms involved in cancer invasion and metastasis. Additionally, microRNA dysregulation contributes significantly to cancer progression. In this study, we aimed to explore the function of miR-503 in cancer metastasis. METHODS: Molecular manipulations (silencing or overexpression) were performed to investigate the biological functions of miR-503 including migration and invasion. Reorganization of cytoskeleton was assessed using immunofluorescence and the relationship between miR-503 and downstream protein tyrosine kinase 7 (PTK7) was assessed using quantitative real-time PCR, immunoblotting, and reporter assays. The tail vein metastatic animal experiments were performed. RESULTS: Herein, we demonstrated that the downregulation of miR-503 confers an invasive phenotype in lung cancer cells and provided in vivo evidence that miR-503 significantly inhibits metastasis. We found that miR-503 inversely regulates EMT, identified PTK7 as a novel miR-503 target, and showed the functional effects of miR-503 on cell migration and invasion were restored upon reconstitution of PTK7 expression. As PTK7 is a Wnt/planar cell polarity protein crucial for collective cell movement, these results implicated miR-503 in both EMT and collective migration. However, the expression of PTK7 did not influence EMT induction, suggesting that miR-503 regulates EMT through mechanisms other than PTK7 inhibition. Furthermore, we discovered that PTK7 mechanistically activates focal adhesion kinase (FAK) and paxillin, thereby controlling the reorganization of the cortical actin cytoskeleton. CONCLUSION: Collectively, miR-503 is capable of governing EMT and PTK7/FAK signaling independently to control the invasion and dissemination of lung cancer cells, indicating that miR-503 represents a pleiotropic regulator of cancer metastasis and hence a potential therapeutic target for lung cancer.

Slug confers resistance to the epidermal growth factor receptor tyrosine kinase inhibitor.

RATIONALE: Non-small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown. OBJECTIVES: To examine the role of EMT regulators in resistance to gefitinib. METHODS: The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription-polymerase chain reaction and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from 44 patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: Slug expression, but not that of snail, twist, or zeb-1, was significantly increased in PC9/gef compared with PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib-induced apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim up-regulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naive cancer cells. CONCLUSIONS: Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

Genomic sequencing and analyses of Lymantria xylina multiple nucleopolyhedrovirus.

BACKGROUND: Outbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylina larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed. RESULTS: The genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hrs) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV. CONCLUSION: The gene parity plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that LyxyMNPV is a Group II NPV that is most closely related to LdMNPV but with a highly distinct genomic organisation.

miR-146b-5p Enhances the Sensitivity of NSCLC to EGFR Tyrosine Kinase Inhibitors by Regulating the IRAK1/NF-κB Pathway.

Although patients with non-small cell lung cancer harboring activating mutations in the epidermal growth factor receptor (EGFR) show good clinical response to EGFR tyrosine kinase inhibitors (TKIs), patients eventually develop acquired resistance. Previous studies have shown that several microRNAs (miRNAs) are involved in EGFR TKI resistance. Here, we aimed to investigate whether miR-146b-5p sensitizes the EGFR TKI-resistant lung cancer cells. Clinical analysis showed that miR-146b-5p expression in lung cancer cells isolated from pleural effusions of treatment-naive patients was significantly higher than that after acquiring resistance to EGFR TKI treatment. Ectopic expression of miR-146b-5p in EGFR TKI-resistant cells enhanced EGFR TKI-induced apoptosis. The same results were observed in EGFR-dependent and -independent osimertinib-resistant primary cancer cells (PE3479 and PE2988). Mechanically, miR-146b-5p suppressed nuclear factor κB (NF-κB) activity and NF-κB-related IL-6 and IL-8 production by targeting IRAK1. A negative correlation was observed between miR-146b-5p and IRAK1 in clinical specimens. In rescue experiments, restoration of IRAK1 expression reversed the effects of miR-146b-5p on EGFR TKI sensitivity and recovered NF-κB-regulated IL-6 and IL-8 production. In conclusion, miR-146b-5p/IRAK1/NF-κB signaling is important in promoting EGFR TKI resistance, and miR-146b-5p may be a useful tool for overcoming EGFR TKI resistance.

MiR-200c-3p suppression is associated with development of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer via a mediating epithelial-to-mesenchymal transition (EMT) process.

BACKGROUND: EGFR-mutant lung cancer inevitably develops resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). OBJECTIVE: To investigate the clinical relevance of microRNAs (miRNAs) in TKI therapy response and resistance. METHODS: We performed a miRNA PCR array analysis and used The Cancer Genome Atlas (TCGA) database to identify potential miRNAs related to EGFR TKIs resistance. We then correlated miRNA expression in 70 surgical and 50 malignant pleural effusion specimens with patient outcomes in those with non-small cell lung carcinoma. Molecular manipulation was performed in EGFR mutant lung cancer cells to assess the effect of miR-200c-3p on cell migratory ability and EGFR-TKI sensitivity. RESULTS: We identified miR-200c-3p and miR-203a-3p as potential EGFR TKI resistance regulators via their modulation of epithelial-to-mesenchymal transition (EMT). MiR-200c-3p and miR-203a-3p were down-regulated in EGFR TKI-resistant cell lines. Progression-free survival (PFS) with EGFR-TKI treatment of patients with high miR-200c-3p expression, but not miR-203a-3p, in the specimens was significantly longer than that of patients with low expression. MiR-200c-3p overexpression inhibited the EMT process in EGFR TKI resistance cell lines and promoted cell death. MiR-200c-3p silencing in EGFR TKI sensitive cell lines increased drug resistance. CONCLUSION: MiR-200c-3p plays a role in sensitivity to EGFR TKIs via modulating EMT process.

Synergistic activation of the tumor suppressor, HLJ1, by the transcription factors YY1 and activator protein 1.

HLJ1 is a novel tumor and invasion suppressor that inhibits tumorigenesis and cancer metastasis. However, the mechanism of HLJ1 activation is currently unclear. Here, we identify an enhancer segment in the HLJ1 gene at -2,125 to -1,039 bp upstream of the transcription start site. A 50-bp element between -1,492 and -1,443 bp is the minimal enhancer segment, which includes the activator protein 1 (AP-1) site (-1,457 to -1,451 bp), an essential regulatory domain that binds the transcriptional factors FosB, JunB, and JunD. Chromatin immunoprecipitation assays confirm that these AP-1 family members bind to a specific site in the HLJ1 enhancer segment in vivo. Overexpression of either YY1 at promoter or AP-1 at enhancer results in a 3-fold increase in the transcriptional activity of HLJ1. We propose a novel mechanism whereby expression of the tumor suppressor, HLJ1, is up-regulated via enhancer AP-1 binding to promoter YY1 and the coactivator, p300, through DNA bending and multiprotein complex formation. The combined expression of AP-1 and YY1 enhances HLJ1 expression by more than five times and inhibits in vitro cancer cell invasion. Elucidation of the regulatory mechanism of HLJ1 expression may facilitate the development of personalized therapy by inhibiting cancer cell proliferation, angiogenesis, and metastasis.

IGFBP7 Drives Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibition in Lung Cancer.

Patients with epidermal growth factor receptor (EGFR) mutation-positive lung cancer show a dramatic response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired drug resistance eventually develops. This study explored the novel mechanisms related to TKI resistance. To identify the genes associated with TKI resistance, an integrative approach was used to analyze public datasets. Molecular manipulations were performed to investigate the roles of insulin-like growth factor binding protein 7 (IGFBP7) in lung adenocarcinoma. Clinical specimens were collected to validate the impact of IGFBP7 on the efficacy of EGFR TKI treatment. IGFBP7 mRNA expression in cancer cells isolated from malignant pleural effusions after acquired resistance to EGFR-TKI was significantly higher than in cancer cells from treatment-naïve effusions. IGFBP7 expression was markedly increased in cells with long-term TKI-induced resistance compared to in TKI-sensitive parental cells. Reduced IGFBP7 in TKI-resistant cells reversed the resistance to EGFR-TKIs and increased EGFR-TKI-induced apoptosis by up-regulating B-cell lymphoma 2 interacting mediator of cell death (BIM) and activating caspases. Suppression of IGFBP7 attenuated the phosphorylation of insulin-like growth factor 1 receptor (IGF-IR) and downstream protein kinase B (AKT) in TKI-resistant cells. Clinically, higher serum IGFBP7 levels and tumors with positive IGFBP7-immunohistochemical staining were associated with poor TKI-treatment outcomes. IGFBP7 confers resistance to EGFR-TKIs and is a potential therapeutic target for treating EGFR-TKI-resistant cancers.

Good response to gefitinib in lung adenocarcinoma of complex epidermal growth factor receptor (EGFR) mutations with the classical mutation pattern.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations are usually detected in lung adenocarcinoma and are associated with a response to EGFR tyrosine kinase inhibitors (TKIs). However, not all EGFR mutations have similarly high clinical response rates. This study aimed to investigate the clinical characteristics and response to gefitinib in lung adenocarcinoma patients with complex EGFR mutations. MATERIALS AND METHODS: Three hundred thirty-nine specimens of lung adenocarcinoma from patients treated with gefitinib were collected for EGFR sequencing. Nineteen patients with complex EGFR mutations were enrolled for the study after excluding three patients with the EGFR T790M mutation, which confers resistance to gefitinib. RESULTS: Among the 19 patients, 12 had complex mutations with the classical mutation pattern (L858R or deletion in exon 19). When compared with those without the classical mutation pattern, patients with this mutation pattern had a higher response rate (83% versus 29%), longer progression-free survival duration (median, 12.7 months versus 4.9 months), and longer overall survival time (median, 24.7 months versus 12.3 months) after gefitinib treatment. Comparing patients harboring complex EGFR mutations with a classical mutation pattern with those harboring single classical mutations, there were no statistical differences in the response rate (83% versus 73%), progression-free survival time (median, 12.7 months versus 8.1 months,) or overall survival time (median, 24.7 months versus 16.4 months). CONCLUSION: Patients with complex EGFR mutations with the classical mutation pattern had the same response rate, progression-free survival duration, and overall survival time as those with single classical mutations. EGFR TKIs may be the choice of treatment for this type of lung adenocarcinoma.

Oncogenic Function of a KIF5B-MET Fusion Variant in Non-Small Cell Lung Cancer.

A kinesin family member 5b (KIF5B)-MET proto-oncogene, receptor tyrosine kinase (MET) rearrangement was reported in patients with lung adenocarcinoma but its oncogenic function was not fully evaluated. We used one-step reverse transcription-polymerase chain reaction for RNA samples to screen for the KIF5B-MET fusion in 206 lung adenocarcinoma and 28 pulmonary sarcomatoid carcinoma patients. Genomic breakpoints of KIF5B-MET were determined by targeted next-generation sequencing. Soft agar colony formation assays, proliferation assays, and a xenograft mouse model were used to investigate its oncogenic activity. In addition, specific MET inhibitors were administered to evaluate their anti-tumor activities. A KIF5B-MET fusion variant in a patient with a mixed-type adenocarcinoma and sarcomatoid tumor was identified, and another case was found in a pulmonary sarcomatoid carcinoma patient. Both cases carried the same chimeric gene, a fusion between exons 1-24 of KIF5B and exons 15-21 of MET. KIF5B-MET-overexpressing cells exhibited significantly increased proliferation and colony-forming ability. Xenograft tumors harboring the fusion gene demonstrated significantly elevated tumor growth. Ectopic expression of the fusion gene stimulated the phosphorylation of KIF5B-MET as well as downstream STAT3, AKT, and ERK1/2 signaling pathways. The MET inhibitors significantly repressed cell proliferation; phosphorylation of downstream STAT3, AKT, and ERK1/2; and xenograft tumorigenicity. In conclusion, the KIF5B-MET variant was demonstrated to have an oncogenic function in cancer cells. These findings have immediate clinical implications for the targeted therapy of subgroups of non-small cell lung cancer patients.