The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

Mycobacterium manresensis induces trained immunity in vitro.

The COVID-19 pandemic posed a global health crisis, with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants weakening vaccine-driven protection. Trained immunity could help tackle COVID-19 disease. Our objective was to analyze whether heat-killed Mycobacterium manresensis (hkMm), an environmental mycobacterium, induces trained immunity and confers protection against SARS-CoV-2 infection. To this end, THP-1 cells and primary monocytes were trained with hkMm. The increased secretion of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, and IL-10, metabolic activity, and changes in epigenetic marks suggested hkMm-induced trained immunity in vitro. Healthcare workers at risk of SARS-CoV-2 infection were enrolled into the MANRECOVID19 clinical trial (NCT04452773) and were administered Nyaditum resae (NR, containing hkMm) or placebo. No significant differences in monocyte inflammatory responses or the incidence of SARS-CoV-2 infection were found between the groups, although NR modified the profile of circulating immune cell populations. Our results show that M. manresensis induces trained immunity in vitro but not in vivo when orally administered as NR daily for 14 days.

Macrophage CD5L is a target for cancer immunotherapy.

BACKGROUND: Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. METHODS: Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. FINDINGS: Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p = 0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages in vitro. Its administration in vivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. INTERPRETATION: CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. FUNDING: For a full list of funding bodies, please see the Acknowledgements.

Macrophages as a Therapeutic Target in Metastatic Prostate Cancer: A Way to Overcome Immunotherapy Resistance?

Prostate cancer (PC) is the most common malignancy and the fifth cause of cancer death in men. The treatment for localized or locally advanced stages offers a high probability of cure. Even though the therapeutic landscape has significantly improved over the last decade, metastatic PC (mPC) still has a poor prognosis mainly due to the development of therapy resistance. In this context, the use of immunotherapy alone or in combination with other drugs has been explored in recent years. However, T-cell directed immune checkpoint inhibitors (ICIs) have shown limited activity with inconclusive results in mPC patients, most likely due to the highly immunosuppressive PC tumor microenvironment (TME). In this scenario, targeting macrophages, a highly abundant immunosuppressive cell type in the TME, could offer a new therapeutic strategy to improve immunotherapy efficacy. In this review, we summarize the growing field of macrophage-directed immunotherapies and discuss how these could be applied in the treatment of mPC, focusing on their combination with ICIs.

Multifaceted Roles of CD5L in Infectious and Sterile Inflammation.

CD5L, a protein expressed and secreted mainly by macrophages, is emerging as a critical immune effector. In addition to its well-defined function as an anti-apoptotic protein, research over the last decade has uncovered additional roles that range from pattern recognition to autophagy, cell polarization, and the regulation of lipid metabolism. By modulating all these processes, CD5L plays a key role in highly prevalent diseases that develop by either acute or chronic inflammation, including several infectious, metabolic, and autoimmune conditions. In this review, we summarize the current knowledge of CD5L and focus on the relevance of this protein during infection- and sterile-driven inflammatory pathogenesis, highlighting its divergent roles in the modulation of inflammation.

ZEB1 promotes inflammation and progression towards inflammation-driven carcinoma through repression of the DNA repair glycosylase MPG in epithelial cells.

OBJECTIVE: Chronic inflammation is a risk factor in colorectal cancer (CRC) and reactive oxygen species (ROS) released by the inflamed stroma elicit DNA damage in epithelial cells. We sought to identify new drivers of ulcerative colitis (UC) and inflammatory CRC. DESIGN: The study uses samples from patients with UC, mouse models of colitis and CRC and mice deficient for the epithelial-to-mesenchymal transition factor ZEB1 and the DNA repair glycosylase N-methyl-purine glycosylase (MPG). Samples were analysed by immunostaining, qRT-PCR, chromatin immunoprecipitation assays, microbiota next-generation sequencing and ROS determination. RESULTS: ZEB1 was induced in the colonic epithelium of UC and of mouse models of colitis. Compared with wild-type counterparts, Zeb1-deficient mice were partially protected from experimental colitis and, in a model of inflammatory CRC, they developed fewer tumours and exhibited lower levels of DNA damage (8-oxo-dG) and higher expression of MPG. Knockdown of ZEB1 in CRC cells inhibited 8-oxo-dG induction by oxidative stress (H2O2) and inflammatory cytokines (interleukin (IL)1ß). ZEB1 bound directly to the MPG promoter whose expression inhibited. This molecular mechanism was validated at the genetic level and the crossing of Zeb1-deficient and Mpg-deficient mice reverted the reduced inflammation and tumourigenesis in the former. ZEB1 expression in CRC cells induced ROS and IL1ß production by macrophages that, in turn, lowered MPG in CRC cells thus amplifying a positive loop between both cells to promote DNA damage and inhibit DNA repair. CONCLUSIONS: ZEB1 promotes colitis and inflammatory CRC through the inhibition of MPG in epithelial cells, thus offering new therapeutic strategies to modulate inflammation and inflammatory cancer.

Tumor-associated macrophages (TAMs) depend on ZEB1 for their cancer-promoting roles.

Accumulation of tumor-associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro-tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial-to-mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor-promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80low pro-tumor phenotype, including direct activation of Ccr2 In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem-like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs' tumor-promoting functions.

ZEB1-induced tumourigenesis requires senescence inhibition via activation of DKK1/mutant p53/Mdm2/CtBP and repression of macroH2A1.

OBJECTIVE: Understand the role of ZEB1 in the tumour initiation and progression beyond inducing an epithelial-to-mesenchymal transition. DESIGN: Expression of the transcription factor ZEB1 associates with a worse prognosis in most cancers, including colorectal carcinomas (CRCs). The study uses survival analysis, in vivo mouse transgenic and xenograft models, gene expression arrays, immunostaining and gene and protein regulation assays. RESULTS: The poorer survival determined by ZEB1 in CRCs depended on simultaneous high levels of the Wnt antagonist DKK1, whose expression was transcriptionally activated by ZEB1. In cancer cells with mutant TP53, ZEB1 blocked the formation of senescence-associated heterochromatin foci at the onset of senescence by triggering a new regulatory cascade that involves the subsequent activation of DKK1, mutant p53, Mdm2 and CtBP to ultimately repress macroH2A1 (H2AFY). In a transgenic mouse model of colon cancer, partial downregulation of Zeb1 was sufficient to induce H2afy and to trigger in vivo tumour senescence, thus resulting in reduced tumour load and improved survival. The capacity of ZEB1 to induce tumourigenesis in a xenograft mouse model requires the repression of H2AFY by ZEB1. Lastly, the worst survival effect of ZEB1 in patients with CRC ultimately depends on low expression of H2AFY and of senescence-associated genes. CONCLUSIONS: The tumourigenic capacity of ZEB1 depends on its inhibition of cancer cell senescence through the activation of a herein identified new molecular pathway. These results set ZEB1 as a potential target in therapeutic strategies aimed at inducing senescence.