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Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes.

ABSTRACT: Chronic exposure to arsenic induces a variety of cancers, particularly in the skin. Autophagy is a highly conserved process which plays a dual role in tumorigenesis. In the present study, we found that chronic exposure to an environmentally relevant dose of arsenite induced malignant transformation of human keratinocytes (HaCaT) with dysregulated autophagy as indicated by an increased number of autophagosomes, activation of mTORC1 pathway, and elevated protein levels of p62 and LC3II. Meanwhile, arsenite-transformed cells showed lower intracellular levels of reactive oxygen species compared with control. Silencing p62 ameliorated elevation in mRNA levels of NRF2 downstream genes (AKR1C1 and NQO1) and malignant phenotypes (acquired invasiveness and anchor-independent growth) induced by chronic arsenite exposure. On the other hand, silencing NRF2 abrogated the increase in mRNA and protein levels of p62 and malignant phenotypes induced by arsenite. In response to acute arsenite exposure, impaired autophagic flux with an increase in p62 protein level and interrupted autophagosome-lysosome fusion was observed. The increase in p62 protein levels in response to arsenite was not completely dependent on NRF2 activation and at least partially attributed to protein degradation. Our data indicate that accumulation of p62 by impaired autophagic flux is involved in the activation of NRF2 and contributes to skin tumorigenesis due to chronic arsenite exposure.

SUBMITTER: Wu X

PROVIDER: S-EPMC6875345 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes.

Wu Xiafang X Sun Ru R Wang Huihui H Yang Bei B Wang Fang F Xu Hongtao H Chen Shimin S Zhao Rui R Pi Jingbo J Xu Yuanyuan Y

Oxidative medicine and cellular longevity 20191030

Chronic exposure to arsenic induces a variety of cancers, particularly in the skin. Autophagy is a highly conserved process which plays a dual role in tumorigenesis. In the present study, we found that chronic exposure to an environmentally relevant dose of arsenite induced malignant transformation of human keratinocytes (HaCaT) with dysregulated autophagy as indicated by an increased number of autophagosomes, activation of mTORC1 pathway, and elevated protein levels of p62 and LC3II. Meanwhile, ...[more]

PMID: 31781319

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Project description:BackgroundInorganic arsenic (iAs) is an environmental toxicant associated with an increased risk of prostate cancer in chronically exposed populations worldwide. However, the biological mechanisms underlying iAs-induced prostate carcinogenesis remain unclear.ObjectivesWe studied how iAs affects normal human prostate stem-progenitor cells (PrSPCs) and drives transformation and interrogated the molecular mechanisms involved.MethodsPrSPCs were enriched by spheroid culture from normal human primary or immortalized prostate epithelial cells, and their differentiation capability was evaluated by organoid culture. Microarray analysis was conducted to identify iAs-dysregulated genes, and lentiviral infection was used for stable manipulation of identified genes. Soft agar colony growth assays were applied to examine iAs-induced transformation. For in vivo study, PrSPCs mixed with rat urogenital sinus mesenchyme were grafted under the renal capsule of nude mice to generate prostatelike tissues, and mice were exposed to 5 ppm (∼65μM) iAs in drinking water for 3 months.ResultsLow-dose iAs (1μM) disturbed PrSPC homeostasis in vitro, leading to increased self-renewal and suppressed differentiation. Transcriptomic analysis indicated that iAs activated oncogenic pathways in PrSPCs, including the KEAP1-NRF2 pathway. Further, iAs-exposed proliferative progenitor cells exhibited NRF2 pathway activation that was sustained in their progeny cells. Knockdown of NRF2 inhibited spheroid formation by driving PrSPC differentiation, whereas its activation enhanced spheroid growth. Importantly, iAs-induced transformation was suppressed by NRF2 knockdown. Mechanistically, iAs suppressed Vacuolar ATPase subunit VMA5 expression, impairing lysosome acidification and inhibiting autophagic protein degradation including p62, which further activated NRF2. In vivo, chronic iAs exposure activated NRF2 in both epithelial and stroma cells of chimeric human prostate grafts and induced premalignant events.ConclusionsLow-dose iAs increased self-renewal and decreased differentiation of human PrSPCs by activating the p62-NRF2 axis, resulting in epithelial cell transformation. NRF2 is activated by iAs through specific autophagic flux blockade in progenitor cells, which may have potential therapeutic implications. https://doi.org/10.1289/EHP6471.

Project description:BackgroundArsenic is a carcinogen that targets the urogenital system, including the prostate. Although the mechanisms for arsenic-induced carcinogenesis are undefined, arsenic drives overaccumulation of stem cells and cancer stem cells (CSCs) in vivo and in vitro, indicating that these cells are a key target population. Disruption of stem cell population dynamics may be critical to acquisition of cancer phenotype. We tested the hypothesis that prostate stem cells have a survival selection advantage during arsenic exposure that favors their accumulation and facilitates their malignant transformation.MethodsInnate and acquired resistance to acute (24-72 hours of exposure) and chronic (6 weeks of exposure) arsenite-induced cytolethality and apoptosis were assessed in a human prostate stem cell line (WPE-stem) and the mature parental cell line (RWPE-1). Real-time reverse transcription-polymerase chain reaction and/or Western blot analysis was used to measure the expression of apoptosis-, stress-, and arsenic-related genes. Arsenic-, cadmium-, and N-methyl-N-nitrosourea-induced isogenic malignant transformants of RWPE-1 cells were compared for acquisition of CSC-like qualities by holoclone and sphere formation assays, growth in soft agar, and expression of CSC biomarkers. All statistical tests were two-sided.ResultsWPE-stem cells showed innate resistance to arsenic-induced cytolethality (arsenite concentration lethal to 50% of the cells [LC(50)] = 32.4 microM, 95% confidence interval [CI] = 31.5 to 33.3 muM) and apoptosis compared with parental RWPE-1 cells (LC(50) = 10.4 muM, 95% CI = 7.4 to 13.4 microM). Compared with RWPE-1 cells, WPE-stem cells showed noticeably higher expression of antiapoptotic (ie, BCL2, MT), stress-related (ie, NFE2L2, SOD1, PRODH), and arsenic adaptation (ie, ABCC1, GSTP1) factors and noticeably lower expression of proapoptotic factors (ie, BAX, caspases 3, 7, 8, and 9). WPE-stem cells also showed hyper-adaptability to chronic arsenite exposure (5 microM, 6 weeks) compared with RWPE-1 cells (LC(50) = 94.7 vs 32.1 microM, difference = 62.6 muM, 95% CI = 53.3 to 71.9 muM) at levels that in previous work induced a malignant phenotype in RWPE-1 after 30 weeks of exposure. Quantification of CSC-like cells in isogenic RWPE-1 transformants showed that marked overproduction was unique to a malignant phenotype acquired in response to arsenic exposure but not in response to cadmium or N-methyl-N-nitrosourea exposure.ConclusionsAn apparent stem cell survival advantage with regard to arsenic causes selection during malignant transformation that manifests itself as an overabundance of CSC-like cells specifically after arsenic-driven acquisition of malignant phenotype. The increased resistance to apoptosis and arsenite hyper-adaptability of WPE-stem cells suggests that arsenite transformation of RWPE-1 cells involves an increase in the number of CSC-like cells.

Dataset Information

Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes.

Publications

Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes.

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