Myeloid-derived Suppressor Cell Promotes The Stem Cell-like Acquisition Of Lung Adenocarcinoma Through Wnt/β-catenin Pathway

Background and Objective:Lung cancer（LC）is the leading cause of cancer-related mortality worldwide^[1,2].Adenocarcinoma is one of the most common subtypes of LC based on histologic features.Despite 20%of early stage patients received surgery,up to 46%of cases developed recurrence or distant metastasis in five years,which serve as a rate-limiting step during the progression^[3].In contrast to the long-held view that tumor recurrence and dissemination occur only at late tumor stages,recent preclinical studies of various cancers have suggested that it could also occur before the onset of clinical symptoms.Accumulating evidence provided more insights into the heterogeneity of cancer and suggested that different gene expression signatures of prognosis can be observed at separate regions of the same tumor.Survive stress factors such as surgery and hypoxia may trigger the switch of remaining tumor cells to a stem cell-like phenotype.The exact identity of cancer stem cell（CSC）still under controversy mainly because of the lack of unique marker and kinetic changes of phenotype.Several attractive candidates have been proposed on the basis of biological behaviors.Side population（SP）in human non-small cell lung carcinoma（NSCLC）,a subpopulation of CSC characterized by their ability to efflux Hoechst 33342 dye,can express ATP-binding cassette transporters to promote drug resistance^[4].High levels of telomerase and low levels of proliferation-related markers were identical to the quiescent feature of stem cell^[4,5].CD133-expressing（CD133⁺）lung cancer cells were capable of regeneration and differentiation into unlimited progeny^[6].CD44⁺lung cancer cells obtained from NSCLC were more indolent than CD44^-cells and exhibited CSC properties such as regeneration in vivo and in vitro^[7].It seems that a minority of lung cancer cells with a high activity of aldehyde dehydrogenase 1（ALDH1）exhibited stem cell-like properties such as proliferation,self-renewal,and multipotential capability^[8].P63 a member of the p53 protein family,is consistently expressed by basal/somatic stem cells.Previous studies have demonstrated that p63 is essential for maintaining of progenitor cells pools^[9].The ectodermal surfaces of the limb buds,branchial arches,and epidermal appendages express p63 during normal development^[12].The deficiency of p63 in transgenic mice models resulted in defects in epithelial development and morphogenesis^[11,12].The role of p63 in the tumorigenesis and progression is still debated.Several studies support the hypothesis that p63 severs as a tumor suppressor.Upregulated p63 contributed to cell cycle arrest and apoptosis^[13,14].P63 genomic sequence was amplified in the majority of lung squamous cell carcinoma（SCC）and endowed patients with prolonged survival[15].Substantial evidence also supports that p63 could function as an oncogene.The dysregulation of p63 was involved in the progression of preneoplastic lesions and lung adenocarcinoma（LAC）^[16,17].Thus,it seems that the crosstalk between p63 and other risk factors can influence the neoplastic progression.Several pathways have been linked to stem cell phenotypes in which p63 is involved.Wnt/β-catenin signaling kept the balance between the proliferation and differentiation of the distal airway epithelium during normal lung development^[18].Activation of canonical Wnt signaling promoted the expansion of bronchioalveolar stem cell^[19].Conditional stabilization ofβ-catenin can inhibit the differentiation of Clara cells and maintain a steady-state pool of self-renewing stem cells^[20].The downregulation of endogenous p63 enhanced the expression of Wnt-related genes^[21].However,the exact relationship between p63 and Wnt/β-catenin during the carcinogenesis and progression of LAC is largely unknown.Cellular sources of Wnt ligands are still unclear.In the setting of hemostasis or tumor,the components of cellular niche such as fibroblast and inflammatory cells have been proposed as attractive candidates.Immune cells,widely appreciated as one of the most important regulators of microenvironment,appear to be at the convergence of the sequential neoplastic progression of lung from non-resolving inflammation to metastasis.NSCLC secreted high levels of chemokines to recruit myeloid-derived suppressor cells（MDSCs）consisting of two major subtypes:polymorphonuclear MDSCs(PMN-MDSCs,CD11b⁺Ly6G⁺Ly6C^lo)and monocytic MDSCs(M-MDSCs,CD11b⁺Ly6G^-Ly6C^hi)^[22].An increased number of PMN-MDSCs was observed in patients with NSCLC compared with the general population^[23].The expansion of PMN-MDSCs has been linked to a relatively poor outcome in NSCLC patients at late stages,suggesting their pro-cancerous effects^[24].Our recent study indicated that histidine decarboxylase（Hdc）-expressing hematopoietic stem cells/progenitor cells（MB-HSC/HSPC）in the bone marrow are myeloid-biased^[25].They may serve as the cellular source of tumor-associated immature myeloid cells in the context of aging or tumor.Hdc⁺immature myeloid cells were involved in the colonic carcinogenesis through direct or indirect effects on parenchymal cells^[10,26].Myeloid-derived Wnts play pivotal roles in the regeneration and differentiation of intestinal stem cells^[27].In the current study,we explored the prognostic role and regulatory mechanisms of p63.Clinical data showed that a minority of patients with early LAC displayed immunopositivity for p63 and exhibited a tendency towards recurrence or metastasis.Hdc⁺PMN-MDSCs were recruited and infiltrated around p63⁺cancer cells.Upregulated canonical Wnts in Hdc⁺PMN-MDSCs aberrantly activated Wnt/β-catenin signaling on cancer cells and promoted p63 expression.Our data further unveil interactions between Hdc⁺PMN-MDSCs and LAC cells,raising the possibility that the inhibition or depletion of these cells with targeted strategies might facilitate anti-lung cancer therapies.Methods:1.The archival blocks of patients with LAC were collected and underwent tissue microarray（TMA）.Serial sections were evaluated by H&E and immunohisto-chemical stains for p63.2.LAC murine models were established using Hdc-CreER^T2;eGFP;LSL-Kras^G12D transgenic mice.Immunohistochemical stains and FACS were employed to explore the number and percentage of MDSCs in malignant tissues.3.The translocation ofβ-catenin in metastatic LAC cells were observed by immunohistochemical stains to evaluated the situation of Wnt/β-catenin signaling.4.Hdc⁺PMN-MDSCs or Hdc⁺PMN-MDSCs-derived Wnts were deleted or inibhited by porcupine^flox/floxlox/flox or iDTR to explore the role of PMN-MDSCs-derived Wnts during the tumorigenesis and progression of LAC,p63 expression,and aberrant activation of Wnt/β-catenin signaling.Results:1.Of 256 patients with early LAC,46 cases（18.0%）exhibited strong and diffuse positivity for p63.2.An increased number of CD15⁺PMN-MDSCs was observed in p63⁺tissues of LAC patients.Immunohistochemical stains and Fluorescence Activated Cell Sorter（FACS）were employed to demonstrate the significant tendency of Hdc⁺PMN-MDSCs towards p63⁺tumor tissues.3.RNA-Seq and RT-PCR results suggested that Hdc⁺PMN-MDSCs obtained from p63⁺tumors expressed high levels of Wnts,including Wnt1,2,3,and 8a.4.In vivo experiments via transgenic models ablated Hdc⁺meyloid cells or Hdc⁺meyloid cells-derived Wnts significantly inhibited the progression of LAC and p63positive rate.Conclusion:This study provided preliminary data to support the important role of MDSCs in LAC progression.Hdc⁺PMN-MDSCs were recruited and expanded in p63⁺malignant tissues,followed by the secretion of high levels of Wnts,which aberrantely activated Wnt/β-catenin signaling with a paracrine pattern in p63⁺LAC cells.Our study keeps the perspective of tailed therepies on the basis of targeting Hdc⁺PMN-MDSCs.