Aryl hydrocarbon receptor undergoes autophagy for lysosomal degradation in human non-small cell lung cancer cell line A549
Poster Number
34
Introduction/Abstract
Lung cancer is the leading cause of cancer death worldwide. Exposure to cigarette smoke and environmental pollutants, which include formaldehyde, benzene, dioxin and polycyclic aromatic hydrocarbons (PAHs), is the major cause of lung cancer. Most of these carcinogenic chemicals play a direct biological role by binding to aryl hydrocarbon receptor (AHR), which is a cytosolic transcription factor. Activation of AHR induces transcription of hundreds of target genes involved in tumorigenesis, inflammation, cell proliferation and apoptosis. Therefore, regulation of AHR in lung cancer could provide a novel way to control the magnitude and duration of AHR-mediated gene regulation. Although AHR plays an important role in cancer development, the role of AHR in tumor pathology is complicated and not fully understood yet. A wide variety of tumor types exhibit both high levels of AHR expression and significant constitutive activity. But the role of AHR in lung cancer seems different from other types of cancer since some studies suggested that AHR suppress invasive ability and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. Their research sparks our interest in studying AHR function in lung tumor cells.
We hope to modulate cellular AHR degradation for regulating AHR function in lung cancer. For most intracellular protein degradation, there are two major proteolytic systems: ubiquitin-proteasome pathway and autophagy-lysosome pathway. Autophagy is a process by which proteins are degraded in lysosomes rather than proteasomes. My results showed that cytoplasmic resting AHR is degraded through lysosome not proteasome. A lot of studies are focused on proteasomal degradation of AHR after ligand binding, but lysosomal degradation of AHR hasn’t been uncovered yet. Understanding of how AHR undergoes autophagy for lysosomal degradation in A549 cells reveals a novel mechanism of AHR degradation and provides new therapeutic targets in lung cancer.
Purpose
We hope to modulate the aryl hydrocarbon receptor degradation via autophagy to control the magnitude and duration of AHR-mediated target genes transcription in non-small cell lung cancer cell line A549.
Through biochemical approaches like gene knockdown or knockout, RT-qPCR, Western Blotting, Co-IP and so on, we determined that chloroquine increased the cytoplasmic resting AHR protein levels and AHR transcriptional activity. The unliganded AHR degradation is mediated by LAMP2, which is a core component of chaperone- mediated autophagy (CMA).
Method
The subject of my study is non-small cell lung cancer cell line A549. Most common biochemical approaches are used to investigate the mechanisms. Such as cell culture, lentivirus vector-mediated shRNA for knockdown of specific gene in cells, CRISPR/Cas9 mediated gene editing, transient transfection, western blot, reverse transcription-quantitative PCR and Co-immunoprecipitation.
Usually, biological triplicate is applied in each experiment, and is repeated at least once. GraphPad Prism 7.0 is used for statistical analysis. The plots showed as the means with error bars (means ± SD, n=3~6). Statistical significance of the differences between group means are evaluated by one-way or two-way ANOVA using Tukey or Sidak test for multiple comparisons. Statistical significance is indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and p>0.05 (ns, not significant).
Results
- Inhibition of lysosomal degradation increases AHR protein level and benzo[a]pyrene (BaP) induced AHR target gene cyp1a1 transcription.
- Cellular AHR protein degradation can be triggered by 6-aminonicotinamide (6AN), which is a CMA inducer. This process can also be blocked by lysosome inhibition via chloroquine, not MG132. Moreover, activation of CMA decreased Bap and FICZ induced AHR target gene cyp1a1 transcription.
- Knockdown of lysosome- associated membrane glycoprotein 2 (LAMP2) increased AHR protein levels and alleviated chloroquine induced AHR accumulation.
- Knockdown of autophagy related 5 (ATG5) in A549 cell line cannot increase AHR protein levels and alleviate chloroquine induced AHR accumulation.
Significance
The degradation of transcription factor AHR upon ligand binding is a critical mechanism in the regulation of AHR mediated target genes transcription. Although this ligand triggered AHR degradation is proteasome dependent, cotreatment with MG132, a 26s proteasome inhibitor, only blocks AHR degradation in a very short time (~2hrs) due to activating macroautophagic degradation of AHR afterwards. However, chloroquine (CQ), as a lysosome inhibitor, provides a new insight to regulate cellular AHR degradation for as long as 24hrs after ligand treatment, which means it can further keep the magnitude and duration of AHR-mediated target genes transcription. I think it is meaningful and significant for clinical application. Some of the AHR ligands are approved by FDA recently, but the degradation of AHR upon ligand binding is still critical in the attenuation of AHR target genes transcription. Combined drug treatment or booster with lysosome inhibitor provides a new way to keep or enhance the effect. In addition, understanding of how AHR undergoes autophagy for lysosomal degradation in A549 cells reveals a novel mechanism of AHR degradation and provides new therapeutic targets in lung cancer. Chaperone mediated autophagy (CMA), as one of the major autophagic pathways for protein degradation, is involved in the degradation of soluble proteins selectively. There are about one third of cytoplasmic proteins which contain KFERQ-like motif are estimated to be CMA substrates. But only a few of them have been identified, especially in the situation that CMA is upregulated in cancer cells for survival. Our previous data showed that AHR can be CMA substrate, and AHR is degraded by CMA inducer 6-aminonicotinamide (6AN). Thus, autophagy inducer can be applied to clinic for preventing environmental pollutants or cigarette smoke, which include formaldehyde, benzene, dioxin and polycyclic aromatic hydrocarbons (PAHs), caused inflammation and lung carcinogenesis through reduced AHR target genes transcription. For example, AHR target gene CYP1a1 metabolizes a number of pro-carcinogens to intermediates that can react with DNA to form adducts, resulting in mutagenesis. In brief, we want to modulate the aryl hydrocarbon receptor degradation via autophagy to control the magnitude and duration of AHR-mediated target genes transcription.
Location
Library and Learning Center, 3601 Pacific Ave., Stockton, CA 95211
Format
Poster Presentation
Aryl hydrocarbon receptor undergoes autophagy for lysosomal degradation in human non-small cell lung cancer cell line A549
Library and Learning Center, 3601 Pacific Ave., Stockton, CA 95211
Lung cancer is the leading cause of cancer death worldwide. Exposure to cigarette smoke and environmental pollutants, which include formaldehyde, benzene, dioxin and polycyclic aromatic hydrocarbons (PAHs), is the major cause of lung cancer. Most of these carcinogenic chemicals play a direct biological role by binding to aryl hydrocarbon receptor (AHR), which is a cytosolic transcription factor. Activation of AHR induces transcription of hundreds of target genes involved in tumorigenesis, inflammation, cell proliferation and apoptosis. Therefore, regulation of AHR in lung cancer could provide a novel way to control the magnitude and duration of AHR-mediated gene regulation. Although AHR plays an important role in cancer development, the role of AHR in tumor pathology is complicated and not fully understood yet. A wide variety of tumor types exhibit both high levels of AHR expression and significant constitutive activity. But the role of AHR in lung cancer seems different from other types of cancer since some studies suggested that AHR suppress invasive ability and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. Their research sparks our interest in studying AHR function in lung tumor cells.
We hope to modulate cellular AHR degradation for regulating AHR function in lung cancer. For most intracellular protein degradation, there are two major proteolytic systems: ubiquitin-proteasome pathway and autophagy-lysosome pathway. Autophagy is a process by which proteins are degraded in lysosomes rather than proteasomes. My results showed that cytoplasmic resting AHR is degraded through lysosome not proteasome. A lot of studies are focused on proteasomal degradation of AHR after ligand binding, but lysosomal degradation of AHR hasn’t been uncovered yet. Understanding of how AHR undergoes autophagy for lysosomal degradation in A549 cells reveals a novel mechanism of AHR degradation and provides new therapeutic targets in lung cancer.
