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.

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.

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.

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.

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.

MicroRNA in Lung Cancer Metastasis.

Tumor metastasis is a hallmark of cancer, with distant metastasis frequently developing in lung cancer, even at initial diagnosis, resulting in poor prognosis and high mortality. However, available biomarkers cannot reliably predict cancer spreading sites. The metastatic cascade involves highly complicated processes including invasion, migration, angiogenesis, and epithelial-to-mesenchymal transition that are tightly controlled by various genetic expression modalities along with interaction between cancer cells and the extracellular matrix. In particular, microRNAs (miRNAs), a group of small non-coding RNAs, can influence the transcriptional and post-transcriptional processes, with dysregulation of miRNA expression contributing to the regulation of cancer metastasis. Nevertheless, although miRNA-targeted therapy is widely studied in vitro and in vivo, this strategy currently affords limited feasibility and a few miRNA-targeted therapies for lung cancer have entered into clinical trials to date. Advances in understanding the molecular mechanism of metastasis will thus provide additional potential targets for lung cancer treatment. This review discusses the current research related to the role of miRNAs in lung cancer invasion and metastasis, with a particular focus on the different metastatic lesions and potential miRNA-targeted treatments for lung cancer with the expectation that further exploration of miRNA-targeted therapy may establish a new spectrum of lung cancer treatments.

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.

Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C.

The epithelial-mesenchymal transition (EMT) regulator, Slug, plays multifaceted roles in controlling lung cancer progression, but its downstream targets and mechanisms in promoting lung cancer progression have not been well defined. In particular, the miRNAs downstream of Slug in non-small cell lung cancer (NSCLC) remain undetermined. Here, we report that miR-137 is downstream of the EMT regulator, Slug, in lung cancer cells. Slug binds directly to the E-box of the miR-137 promoter and up-regulates its expression in lung cancer cells. Knockdown of miR-137 abolished Slug-induced cancer invasion and migration, whereas upregulation of miR-137 was found to trigger lung cancer cell invasion and progression by direct suppressing TFAP2C (transcription factor AP-2 gamma). Clinical data showed that lung adenocarcinoma patients with low-level expression of Slug and miR-137 but high-level expression of TFAP2C experienced significantly better survival. miR-137 is a Slug-induced miRNA that relays the pro-metastatic effects of Slug by targeting TFAP2C. Our findings add new components to the Slug-mediated regulatory network in lung cancer, and suggest that Slug, miR-137, and TFAP2C may be useful prognostic markers in lung adenocarcinoma.

EGFR-L858R mutant enhances lung adenocarcinoma cell invasive ability and promotes malignant pleural effusion formation through activation of the CXCL12-CXCR4 pathway.

Malignant pleural effusion (MPE) is a common clinical problem in non-small cell lung carcinoma (NSCLC) patients; however, the underlying mechanisms are still largely unknown. Recent studies indicate that the frequency of the L858R mutant form of the epidermal growth factor receptor (EGFR-L858R) is higher in lung adenocarcinoma with MPE than in surgically resected specimens, suggesting that lung adenocarcinoma cells harboring this mutation tend to invade the adjacent pleural cavity. The purpose of this study was to clarify the relationship between the EGFR-L858R mutation and cancer cell invasion ability and to investigate the molecular mechanisms involved in the formation of MPE. We found that expression of EGFR-L858R in lung cancer cells resulted in up-regulation of the CXCR4 in association with increased cancer cell invasive ability and MPE formation. Ectopic expression of EGFR-L858R in lung cancer cells acted through activation of ERK signaling pathways to induce the expression of CXCR4. We also indicated that Inhibition of CXCR4 with small interfering RNA, neutralizing antibody, or receptor antagonist significantly suppressed the EGFR-L858R-dependent cell invasion. These results suggest that targeting the production of CXCR4 and blocking the CXCL12-CXCR4 pathway might be effective strategies for treating NSCLCs harboring a specific type of EGFR mutation.

IL-8 confers resistance to EGFR inhibitors by inducing stem cell properties in lung cancer.

Epidermal growth factor receptor (EGFR)-targeted strategy is limited by resistance. We identify the potential genes involved in EGFR TKI (tyrosine kinase inhibitor) resistance and study the therapeutic mechanism in the non-small cell lung cancers. Potential genes involved in resistance were examined by analyzing datasets from a pair of EGFR TKI-sensitive (PC9) and TKI-resistant cells (PC9/gef). Blood specimens from patients taking EGFR TKI as first-line treatment were used to examine the correlation between drug's efficacy and IL-8 level. The effects of IL-8 on gefitinib-induced apoptosis, stemness, and in vivo tumorigenicity were investigated using established cell lines. We identified IL-8 was up-regulated in gefitinib-resistant cells, and high plasma IL-8 level was correlated with shorter progression-free-survival time. IL-8 overexpression suppressed gefitinib-induced apoptosis in gefitinib-sensitive cells. By contrast, suppression of IL-8 enhanced gefitinib-induced cell death in gefitinib-resistant cells. IL-8 also increased stem-like characteristics including aldehyde dehydrogenase activity, expression of stemness-related genes, clonogenic activity, side-population, and in vivo tumorigenicity. Consistently, knockdown of IL-8 leads to loss of stem cell-like characteristics in gefitinib-resistant cells. Our study demonstrates an important role for IL-8, and suggests IL-8 is a potential therapeutic target for overcoming EGFR TKI resistance.

ALDH-positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors.

Molecular targeting therapeutics, such as EGFR tyrosine kinase inhibitors (TKIs), are important treatment strategies for lung cancer. Currently, the major challenge confronting targeted cancer therapies is the development of resistance. Cancer stem cells (CSCs) represent a rare population of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance and spreading. Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs. However, the issue of whether CSCs contribute to EGFR TKI resistance in lung cancer is yet to be established. In the current study, we explored the association of ALDH1A1 expression with EGFR TKI resistance in lung cancer stem cells. ALDH1A1-positive lung cancer cells displayed resistance to gefitinib, compared to ALDH1A1-negative lung cancer cells. Moreover, PC9/gef cells (gefitinib-resistant lung cancer cells) presented a higher proportion of ALDH1A1-positive cells, compared to PC9 cells (gefitinib-sensitive lung cancer cells). Clinical sample studies were consistent with results from cell culture model systems showing that lung cancer cells with resistance to EGFR TKI and chemotherapy drugs contain significantly increased proportions of ALDH1A1-positive cells. These findings collectively suggest that ALDH1A1 positivity in cancer stem cells confers resistance to EGFR TKI in 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.

Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma.

PURPOSE: In a previous genome-wide gene expression profiling analysis using an invasion cancer cell lines model, we have identified Slug as selectively overexpressed in the highly invasive cancer cells. Here, we investigated the clinical significance of Slug in lung adenocarcinoma and the role of Slug in the process of cancer cell invasion and metastasis. EXPERIMENTAL DESIGN: Real-time quantitative reverse transcription-PCR was used to investigate Slug mRNA in surgically resected lung adenocarcinoma of 54 patients and its correlation with survival. We overexpressed Slug in a lung adenocarcinoma cell line with very low Slug levels and investigated the in vitro and in vivo effects of Slug expression. RESULTS: High expression of Slug mRNA in lung cancer tissue was significantly associated with postoperative relapse (P = 0.03) and shorter patient survival (P < 0.001). The overexpression of Slug enhanced xenograft tumor growth and increased microvessel counts in angiogenesis assay. Both inducible and constitutive overexpression of Slug suppressed the expression of E-cadherin and increased the in vitro invasive ability. Zymography revealed increased matrix metalloproteinase-2 activity in Slug overexpressed cells. ELISA, reverse transcription-PCR, and immunohistochemistry confirmed the increase of matrix metalloproteinase-2 proteins and mRNA in Slug overexpressed cells and xenograft tumors. CONCLUSIONS: Slug expression can predict the clinical outcome of lung adenocarcinoma patients. Slug is a novel invasion-promoting gene in lung adenocarcinoma.

Correlation between gene expression of aryl hydrocarbon receptor (AhR), hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1) and 1B1 (CYP1B1), and inducibility of CYP1A1 and CYP1B1 in human lymphocytes.

The relationships between gene expression of aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1), 1B1 (CYP1B1), CYP1A1, and the inducibility of CYP1A1 and CYP1B1 were determined in 32 cultivated human lymphocytes. Cytochrome P450 induction was performed by incubating lymphocytes with benzanthracene. The relative gene expression levels were determined by quantitative real-time RT-PCR assay. We found that gender is an important confounding factor for gene expression in cultivated lymphocytes. AhR, CYP1A1 and CYP1B1 levels in noninduced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers (p < 0.05). Nevertheless, CYP1A1 and CYP1B1 inducibility was lower in female nonsmokers. CYP1A1 inducibility was higher in male smokers than in male nonsmokers (p < 0.05). After controlling for gender and cigarette smoking, AhR levels positively correlated with CYP1B1 levels and CYP1A1 inducibility (p < 0.01 and p = 0.03, respectively). Arnt levels also correlated with CYP1B1 levels in induced lymphocytes (p < 0.01). However, AhR levels were negatively correlated with CYP1B1 inducibility. These data indicate that AhR expression associates with individual variation of CYP1A1 inducibility and CYP1B1 expression in cultivated lymphocytes. Furthermore, gender and cigarette smoking are important confounding factors for gene expression levels in cultivated lymphocytes.