Role of ZEB1 in macrophages during homeostasis, inflammation and cancer

Abstract

[eng] ZEB1 is a transcription factor whose expression in cancer cells promotes tumor initiation and progression. In this study, we for the first time characterized Zeb1 and study its function in macrophages under either homeostasis or activation conditions as well as in a murine cancer model. We found that macrophages deficient for Zeb1 showed aberrant characteristics in phenotype and functions, under physiological and pathological conditions. Here we clarified a functional role of Zeb1 on macrophages playing a role in macrophage phagocytosis, migration and inflammation as well as in tumor progression in a non-cell-autonomous manner modulating the tumor microenviroment. In fact the evidence presented indicates that the downregulation of Zeb1 in macrophages is associated with the inhibition of TAM characteristics and inhibition of tumor progression.

ZEB1 plays important roles during embryogenesis and deletion of both alleles of Zeb1 in mice results in embryonic lethality. ZEB1 represses key genes involved in the terminal differentiation of multiple tissues, including inter alia epithelial cells, pituitary gland, skeletal and smooth muscle, cartilage, and bone. Although ZEB1 is expressed in lymphoid cells where it represses pivotal hematopoietic transcription factors, there was no evidence for a role of ZEB1 in the regulation of lymphoid or myeloid differentiation. We showed here that downregulation of Zeb1 in bone marrow precursors promoted their differentiation towards macrophages. These data further support a model, best characterized in epithelial tissues and skeletal muscle, where ZEB1 expression needs to decline for early precursors to terminally differentiate.

ZEB1 has been extensively characterized in cancer cells where it promotes their stemness, survival and invasiveness. However, its role in the tumor microenvironment remained to be elucidated. Among cancer cells, ZEB1 is not expressed across the entire tumor mass but is rather restricted to a subpopulation of stem-like malignant cells at the invasive front, actually, at the interface where cancer cells and TAMs interact. Although ZEB1 expression among stromal cells has been noted, the identity of the cell types expressing ZEB1 has not been established. This study showed that ZEB1 is also expressed in TAMs and that ZEB1 not only bilaterally regulates the crosstalk between cancer cells and TAMs but that this crosstalk regulates ZEB1 expression itself. Thus, Zeb1 was upregulated in macrophages that have interacted with cancer cells as well as in cancer cells that have interacted with wild-type TAMs. The tumor-promoting role of ZEB1 is therefore supported by a positive feedback of its expression between malignant cells and TAMs. We found that Zeb1 is restricted to the F4/80low macrophage/TAM subpopulation—previously known to display stronger pro-tumor and pro-angiogenic functions—whose share is expanded by ZEB1.

Soluble factors produced by the tumor—e.g., CSF1 and CCL2—attract F4/80low CCR2+ monocytes into their microenvironment where they are activated into TAMs. Inhibition of the CCL2–CCR2 axis blocks monocyte recruitment into the tumor stroma and inhibits tumor growth. We found that Zeb1 promotes monocyte migration both in response to chemotactic stimuli (CSF1 and CCL2) and in the context of cancer. Zeb1-deficient TAMs expressed lower levels of Ccr2 and were unable to induce Ccl2 in ID8 cells. At the same time, the maximum effect of ZEB1 as a biomarker of poorer prognosis in ovarian cancer patients depended on high levels of CCL2. These data establish Zeb1 as an important inducer of the pro-tumor and pro-metastatic CCR2-MMP9-CCL2 loop between tumor cells and TAMs.

It is important to note that this CCR2-MMP9-CCL2 loop was inhibited by just a partial downregulation of Zeb1 in TAMs. Data here showed that the pro-tumor role of ZEB1 in TAMs also depends on a similarly narrow threshold of expression. Zeb1 (+/-) macrophages still express about half of the Zeb1 mRNA levels of wild-type macrophages, but this downregulation was enough to render Zeb1 (+/-) TAMs unable to promote tumor growth when transplanted into tumor-bearing mice as wild-type macrophages did. As in the case of ZEB1 expression in cancer cells, to the best of our knowledge, this is the first example of a heterozygous gene deletion being sufficient to block the tumor-promoting role of TAMs.

Expression of ZEB1 in cancer cells has been associated to increased chemotherapy resistance. In parallel, we found here that expression of ZEB1 in TAMs also increased the cancer cell resistance to chemotherapy. In that line, we showed that Zeb1 in TAMs increased the expression of Il10, Mmp9 and Il1b—that have a suppressor effect on chemotherapy—and of the drug efflux transporter Mdr1. The dual role of ZEB1 promoting tumor progression in cancer cells and in TAMs—albeit through different mechanisms—has translational implications. Targeting ZEB1 in cancer cells is being considered in ongoing clinical trials but data here suggest that improving chemotherapy response would also require the downregulation of ZEB1 in TAMs. The fact that a partial downregulation of Zeb1 in TAMs was sufficient to abolish TAMs’ tumor-promoting function is highly relevant for therapy approaches aiming at blocking ZEB1 expression and/or function.

These results establish a new role for ZEB1 promoting tumor progression through its expression in TAMs, thus setting ZEB1 expression as a relevant target in cancer therapy.