Regulation of Tracheal Stem Cell Injury Repair and the Lung Cancer Microenvironment by UHRF1

Abstract

Epigenetic regulators play pivotal roles in many fundamental biological processes, including cell proliferation, cell death, and cell fate specification. As epigenetic abnormalities are frequently associated with human diseases, it is of great importance to understand the key genes involved in disease etiology. Ubiquitin Like with PHD and Ring Finger Domains 1 (UHRF1) is a multi-domain epigenetic regulator. By working coordinately with DNA methyltransferase 1 (DNMT1), UHRF1 plays a crucial role in maintaining epigenome integrity, especially in heterochromatin regions and retrotransposon elements. Knockout of Uhrf1 in mice is embryonic lethal and thus conditional Uhrf1 knockout mice have been used to delineate the role of UHRF1 in diverse biological contexts. These studies have revealed the essential functions for UHRF1 in regulating the proliferation, survival, and differentiation of colonic regulatory T cells, invariant natural killer T (iNKT) cells, neural stem cells, and hematopoietic stem cells. However, the function of UHRF1 in regulating self-renewal and differentiation in epithelial stem and progenitor cells has not been investigated. Additionally, although UHRF1 expression is upregulated in several types of cancer, most studies have focused on how UHRF1 affects cancer cell-autonomous processes instead of how this molecule impacts the tumor microenvironment in vivo. The airway is lined by a pseudostratified mucociliary epithelium, which is composed of multiple cell types. Airway basal cells function as stem cells to replenish the mucociliary epithelium in response to airway injury. By analyzing a published dataset, we found that Uhrf1 was among the genes most highly upregulated in response to sulfur dioxide-induced airway injury. However, the functional significance of UHRF1 induction for the airway repair process has not been explored. Here we show that UHRF1 expression was mainly restricted to proliferating basal and progenitor cells during the regeneration of the mucociliary epithelium in vivo. In three-dimensional organoid culture, we also found that UHRF1 was expressed in proliferating human bronchial epithelial (HBE) cells. Reduction of UHRF1 expression by shRNAs in HBE cells reduced cell proliferation, an effect that is partially mediated by the cyclin dependent kinase inhibitor p15. To further evaluate the function of UHRF1 in basal cells in vivo, we generated a basal cell-specific Uhrf1-knockout mouse model and found that Uhrf1-null basal cells did not proliferate after airway injury. This proliferation defect and the G1 cell cycle arrest were linked to an inability of cells to enter S phase and form DNA replication complex, as evident by loss of PCNA puncta. Perturbation of DNA methylation is unlikely to contribute to the proliferation defect, since Uhrf1-null basal cells halt cell cycle progression and thus cannot passively loss genome methylation without DNA replication. Our results implicate a new paradigm for UHRF1 in regulating adult epithelial stem and progenitor cells.We next sought to explore the function of UHRF1 in cancer development. Consistent with prior reports, we found that in non-small cell lung cancer (NSCLC) patients, the mRNA level of UHRF1 was markedly upregulated in tumor regions compared to adjacent non-tumor lung tissues. Immunostaining analysis showed that UHRF1 was barely detected in normal lung tissue, whereas its expression was easily detected in the papillary adenocarcinoma in a KRAS G12V-driven spontaneous lung adenocarcinoma mouse model. To investigate the role of UHRF1 in regulating lung tumor growth, we conducted experiments using a syngeneic NSCLC mouse model termed Lewis lung carcinoma (LLC). Knockdown of UHRF1 did not affect cancer cell growth in vitro. Surprisingly, UHRF1 downregulation in tumor cells strongly suppresses tumor growth in immune-competent, but not immune-compromised mice. Higher percentages of tumor-infiltrating CD4+ and CD8+ T cells were observed in UHRF1 knockdown tumors, and the tumor-infiltrating T cells had enhanced proliferative capacity as well as cytotoxicity. Further experimental results described here indicate that the loss of UHRF1 exerts anti-tumor immunity potentially mediated by downregulation of the immune checkpoint protein PD-L1. Taken together, our work provides new insights into the functionality of UHRF1 in controlling the complex process underlying NSCLC tumor growth and emphasizes the necessity for immunocompetent mouse models to study the reciprocal interactions between tumor cells and the microenvironment.