Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

  1. Dan-Dan Zou
  2. Ya-Zhou Sun
  3. Xin-Jie Li
  4. Wen-Juan Wu
  5. Dan Xu
  6. Yu-Tong He
  7. Jue Qi
  8. Ying Tu
  9. Yang Tang
  10. Yun-Hua Tu
  11. Xiao-Li Wang
  12. Xing Li
  13. Feng-Yan Lu
  14. Ling Huang
  15. Heng Long
  16. Li He  Is a corresponding author
  17. Xin Li  Is a corresponding author
  1. First Affiliated Hospital of Kunming Medical University, China
  2. Sun Yat-sen University, China
  3. Shanghai Changzheng Hospital, China
  4. People's Hospital of Chuxiong Yi Autonomous Prefecture, China
  5. Qujing Affiliated Hospital of Kunming Medical University, China
  6. First Affiliated Hospital of Dali University, China
  7. Wenshan Zhuang and Miao Autonomous Prefecture Specialist Hospital of Dermatology, China

Abstract

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent of the keratinocyte-derived malignancies with actinic keratosis (AK) as a precancerous lesion. To comprehensively delineate the underlying mechanisms for the whole progression from normal skin to AK to invasive cSCC, we performed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomes of 138,982 cells from 13 samples of six patients including AK, squamous cell carcinoma in situ (SCCIS), cSCC and their matched normal tissues, covering comprehensive clinical courses of cSCC. We identified diverse cell types, including important subtypes with different gene expression profiles and functions in major keratinocytes. In SCCIS, we discovered the malignant subtypes of basal cells with differential proliferative and migration potential. Differentially expressed genes (DEGs) analysis screened out multiple key driver genes including transcription factors (TFs) along AK to cSCC progression. Immunohistochemistry (IHC) / immunofluorescence (IF) experiments and single-cell ATAC sequencing (scATAC-seq) data verified the expression changes of these genes. The functional experiments confirmed the important roles of these genes in regulating cell proliferation, apoptosis, migration and invasion in cSCC tumor. Furthermore, we comprehensively described the tumor microenvironment (TME) landscape and potential keratinocyte-TME crosstalk in cSCC providing theoretical basis for immunotherapy. Together, our findings provide a valuable resource for deciphering the progression from AK to cSCC and identifying potential targets for anticancer treatment of cSCC.

Data availability

The raw data and gene counts table are available from GEO under accession number (GSE193304). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

The following data sets were generated

Article and author information

Author details

  1. Dan-Dan Zou

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  2. Ya-Zhou Sun

    School of Medical, Sun Yat-sen University, Shenzhen, China
    Competing interests
    The authors declare that no competing interests exist.
  3. Xin-Jie Li

    School of Medical, Sun Yat-sen University, Shenzhen, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Wen-Juan Wu

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  5. Dan Xu

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  6. Yu-Tong He

    School of Medical, Sun Yat-sen University, Shenzhen, China
    Competing interests
    The authors declare that no competing interests exist.
  7. Jue Qi

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  8. Ying Tu

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  9. Yang Tang

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  10. Yun-Hua Tu

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    Competing interests
    The authors declare that no competing interests exist.
  11. Xiao-Li Wang

    Department of Dermatology, Shanghai Changzheng Hospital, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.
  12. Xing Li

    Department of Dermatology, People's Hospital of Chuxiong Yi Autonomous Prefecture, Chuxiong, China
    Competing interests
    The authors declare that no competing interests exist.
  13. Feng-Yan Lu

    Department of Dermatology, Qujing Affiliated Hospital of Kunming Medical University, Qujing, China
    Competing interests
    The authors declare that no competing interests exist.
  14. Ling Huang

    Department of Dermatology, First Affiliated Hospital of Dali University, Dali, China
    Competing interests
    The authors declare that no competing interests exist.
  15. Heng Long

    Wenshan Zhuang and Miao Autonomous Prefecture Dermatology Clinic, Wenshan Zhuang and Miao Autonomous Prefecture Specialist Hospital of Dermatology, Wenshan, China
    Competing interests
    The authors declare that no competing interests exist.
  16. Li He

    Department of Dermatology, First Affiliated Hospital of Kunming Medical University, Kunming, China
    For correspondence
    drheli2662@126.com
    Competing interests
    The authors declare that no competing interests exist.
  17. Xin Li

    School of Medical, Sun Yat-sen University, Shenzhen, China
    For correspondence
    lixin253@mail.sysu.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8328-4894

Funding

Yunnan Science and Technology Leading Talents Project (2017HA010)

  • Li He

National Natural Science Foundation of China (82260517)

  • Xin Li

Yunnan High-level Talents Scientific Research Project (2023-KHRCBZ-B13)

  • Dan-Dan Zou

Yunnan Province Clinical Research Center for Skin Immune Diseases (2019ZF012)

  • Li He

Yunnan Province Clinical Center for Skin Immune Diseases (ZX2019-03-02)

  • Li He

Shenzhen Science and Technology Program (JCYJ20190807160011600)

  • Xin Li

Shenzhen Science and Technology Program (JCYJ20210324124808023)

  • Xin Li

China Postdoctoral Science Foundation (2020M683073)

  • Ya-Zhou Sun

Guangzhou Science Technology Project (201904010007)

  • Xin Li

Guangdong Provincial Key Laboratory of Digestive Cancer Research (2021B1212040006)

  • Xin Li

National Natural Science Foundation of China (81872299)

  • Xin Li

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). This study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University (Approval Number (2020)-L-29), and written informed consent was obtained from all patients.

Copyright

© 2023, Zou et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,799
    views
  • 478
    downloads
  • 14
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Dan-Dan Zou
  2. Ya-Zhou Sun
  3. Xin-Jie Li
  4. Wen-Juan Wu
  5. Dan Xu
  6. Yu-Tong He
  7. Jue Qi
  8. Ying Tu
  9. Yang Tang
  10. Yun-Hua Tu
  11. Xiao-Li Wang
  12. Xing Li
  13. Feng-Yan Lu
  14. Ling Huang
  15. Heng Long
  16. Li He
  17. Xin Li
(2023)
Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma
eLife 12:e85270.
https://doi.org/10.7554/eLife.85270

Share this article

https://doi.org/10.7554/eLife.85270

Further reading

    1. Cancer Biology
    2. Genetics and Genomics
    Hirokazu Kimura, Kamel Lahouel ... Nicholas Jason Roberts
    Research Article

    Interpretation of variants identified during genetic testing is a significant clinical challenge. In this study, we developed a high-throughput CDKN2A functional assay and characterized all possible human CDKN2A missense variants. We found that 17.7% of all missense variants were functionally deleterious. We also used our functional classifications to assess the performance of in silico models that predict the effect of variants, including recently reported models based on machine learning. Notably, we found that all in silico models performed similarly when compared to our functional classifications with accuracies of 39.5–85.4%. Furthermore, while we found that functionally deleterious variants were enriched within ankyrin repeats, we did not identify any residues where all missense variants were functionally deleterious. Our functional classifications are a resource to aid the interpretation of CDKN2A variants and have important implications for the application of variant interpretation guidelines, particularly the use of in silico models for clinical variant interpretation.

    1. Cancer Biology
    2. Developmental Biology
    Sara Jaber, Eliana Eldawra ... Franck Toledo
    Research Article

    Missense ‘hotspot’ mutations localized in six p53 codons account for 20% of TP53 mutations in human cancers. Hotspot p53 mutants have lost the tumor suppressive functions of the wildtype protein, but whether and how they may gain additional functions promoting tumorigenesis remain controversial. Here, we generated Trp53Y217C, a mouse model of the human hotspot mutant TP53Y220C. DNA damage responses were lost in Trp53Y217C/Y217C (Trp53YC/YC) cells, and Trp53YC/YC fibroblasts exhibited increased chromosome instability compared to Trp53-/- cells. Furthermore, Trp53YC/YC male mice died earlier than Trp53-/- males, with more aggressive thymic lymphomas. This correlated with an increased expression of inflammation-related genes in Trp53YC/YC thymic cells compared to Trp53-/- cells. Surprisingly, we recovered only one Trp53YC/YC female for 22 Trp53YC/YC males at weaning, a skewed distribution explained by a high frequency of Trp53YC/YC female embryos with exencephaly and the death of most Trp53YC/YC female neonates. Strikingly, however, when we treated pregnant females with the anti-inflammatory drug supformin (LCC-12), we observed a fivefold increase in the proportion of viable Trp53YC/YC weaned females in their progeny. Together, these data suggest that the p53Y217C mutation not only abrogates wildtype p53 functions but also promotes inflammation, with oncogenic effects in males and teratogenic effects in females.