ZEB2 drives intra-tumor heterogeneity and skin squamous cell carcinoma formation with distinct EMP transition states.
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ABSTRACT: Poor prognosis in cutaneous squamous cell carcinoma (cSCC) stems from poor differentiation, invasion and metastasis, all of which are linked to the process of epithelial-to-mesenchymal plasticity (EMP). Here we showed that the EMP-associated transcription factor ZEB2 drives cSCC heterogeneity which resembles biphasic carcinosarcoma-like tumors. Single cell RNA sequencing revealed different subpopulations ranging from fully epithelial (E) to intermediate (EM) to fully mesenchymal (M) which was associated with the graded loss of cell surface markers EPCAM, CDH1, ITGB4 and CD200. Mesenchymal features were associated with a higher metastatic capacity and anoikis resistance, yet this comes with a sensitivity towards TNF-induced cell death. Altogether we provide insights in cSCC heterogeneity and modes to target mesenchymal- metastasis inducing cells
Project description:The CD200-CD200R immunoregulatory signaling axis plays an etiological role in the survival and spread of numerous cancers primarily through suppression of anti-tumor immune surveillance. Our previous work outlined a pro-metastatic role for the CD200-CD200R axis in cutaneous squamous cell carcinoma (cSCC) that is independent of direct T cell suppression but modulates the function of infiltrating myeloid cells. To identify effectors of the CD200-CD200R axis important for cSCC metastasis, we conducted RNA-Seq profiling of infiltrating CD11b+Cd200r+ cells isolated from Cd200+ versus Cd200 null cSCCs and identified the cysteine protease Cathepsin K (Ctsk) to be highly upregulated in Cd200+ cSCCs. CD11b+Cd200r+ cells, which expressed phenotypic markers associated with myeloid-derived suppressor cell-like cells and tumor-associated macrophages, were the primary source of Ctsk expression in cSCC. Using a Cd200r+ myeloid cell-cSCC co-culture system, we observed that induction of Ctsk in Cd200r+ tumor-infiltrating cells was dependent on engagement of the CD200-CD200R axis, indicating that Ctsk is a target gene of this pathway in the cSCC tumor microenvironment. Inhibition of Ctsk, but not matrix metalloproteinases (MMPs), significantly blocked cSCC cell migration through collagen gels in vitro. Finally, both targeted Cd200 disruption in tumor cells and Ctsk pharmacological inhibition dramatically reduced cSCC metastasis in vivo. Collectively, our findings identify a novel direct role for Cd200r+ infiltrating myeloid lineages in tumor metastasis and support the conclusion that CD200 stimulates cSCC invasion and metastasis via induction of Ctsk in CD200r+ infiltrating cells.
Project description:Sustained tumor progression has been attributed to a distinct population of tumor-propagating cells (TPCs). To identify TPCs relevant to lung cancer pathogenesis, we investigated functional heterogeneity in tumor cells isolated from Kras-driven mouse models of non-small cell lung cancer (NSCLC). CD24+ITGB4+Notchhi cells are capable of propagating tumor growth in both a clonogenic and an orthotopic serial transplantation assay. While all four Notch receptors mark TPCs, Notch3 plays a non-redundant role in tumor cell propagation in two mouse model and in human NSCLC. The TPC population is enriched after chemotherapy and the gene signature of mouse TPCs correlates with poor prognosis in human NSCLC. The unique role of Notch3 in tumor propagation may provide a therapeutic target for NSCLC Primary lung adenocarcinoma tumor cells were FACS sorted based on expression of CD24, ITGB4 and Notch. TPC cells are defined by Cd24+ITGB4+ Notch(high), and the remainder tumor cells are non-TPC cells. Samples were derived from six mice.
Project description:Early cutaneous squamous cell carcinomas (cSCCs) show epithelial differentiation features and good prognosis, whereas advanced cSCCs present mesenchymal traits and are associated with tumor relapse, metastasis, and poor survival. Prognostic biomarkers for cSCC relapse must therefore be found that accurately predict the clinical course of the disease, since established markers are suboptimal. Using mouse models of cSCC progression, we showed that the emergence of epithelial plastic cancer cells with a hybrid epithelial/mesenchymal phenotype promotes tumor progression to a mesenchymal state. These cells can be identified early on by the expression of integrin αV (ITGAV). Analysis of ITGAV expression in a cohort of primary cSCCs from patients provided prognostic information about the risk of relapse beyond current histopathological parameters. Together, our findings provide an opportunity to clinically implement ITGAV by standard immunodetection approaches and thereby improve patient stratification and therapeutic management.
Project description:Analysis of gene expression profiles of matrix-detached cells with and without expression of ITGB4, in clustering and non-clustering conditions. The experiment tested the hypothesis that the integrin beta 4 (ITGB4) mediates a significant amount of pro-survival signaling in matrix-detached conditions. Expression of ITGB4 in cancer is correlated with poor patient survival and is impliated in increased metastatic spread. Survival in matrix-deprived conditions is essential to metastasis and targeting signaling downstream of the integrin beta 4 may help curtail metasasis.
Project description:Background Oral squamous cell carcinoma (OSCC), an HPV-negative head and neck cancer, frequently metastasizes to the regional lymph nodes but only occasionally beyond. Initial phases of metastasis are associated with an epithelial–mesenchymal transition (EMT), while the consolidation phase is associated with mesenchymal–epithelial transition (MET). This dynamic is referred to as epithelial–mesenchymal plasticity (EMP). While it is known that EMP is essential for cancer cell invasion and metastatic spread, less is known about the heterogeneity of EMP states and even less about the heterogeneity between primary and metastatic lesions. Methods To assess both the heterogeneity of EMP states in OSCC cells and their effects on stromal cells, we performed single-cell RNA sequencing (scRNAseq) of 5 primary tumors, 9 matching metastatic and 5 tumor-free lymph nodes and re-analyzed publicly available scRNAseq data of 9 additional primary tumors. For examining the cell type composition, we performed bulk transcriptome sequencing. Protein expression of selected genes were confirmed by immunohistochemistry. Results From the 23 OSCC lesions, the single cell transcriptomes of a total of 7263 carcinoma cells were available for in-depth analyses. We initially focused on one lesion to avoid confounding inter-patient heterogeneity and identified OSCC cells expressing genes characteristic of different epithelial and partial EMT stages. RNA velocity and the increase in inferred copy number variations indicated a progressive trajectory towards epithelial differentiation in this metastatic lesion, i.e., cells likely underwent MET. Extension to all samples revealed a less stringent but essentially similar pattern. Interestingly, MET cells show increased activity of the EMT-activator ZEB1. Immunohistochemistry confirmed that ZEB1 was co-expressed with the epithelial marker cornifin B in individual tumor cells. The lack of E-cadherin mRNA expression suggests this is a partial MET. Within the tumor microenvironment we found immunomodulating fibroblasts that were maintained in primary and metastatic OSCC. Conclusions This study reveals that EMP enables different partial EMT and epithelial phenotypes of OSCC cells, which are endowed with capabilities essential for the different stages of the metastatic process, including maintenance of cellular integrity. During MET, ZEB1 appears to be functionally active, indicating a more complex role of ZEB1 than mere induction of EMT.
Project description:Epithelial to Mesenchymal Transition (EMT) has been associated with cancer cell heterogeneity, plasticity and metastasis. It has been the subject of several modeling effort. This logical model of the EMT cellular network aims to assess microenvironmental signals controlling cancer-associated phenotypes amid the EMT continuum. Its outcomes relate to the qualitative degrees of cell adhesions by adherent junctions and focal adhesions, two features affected during EMT. Model attractors recover epithelial, mesenchymal and hybrid phenotypes, and simulations show that hybrid phenotypes may arise through independent molecular paths, involving stringent extrinsic signals.
Of particular interest, model predictions and their experimental validations indicated that: 1) ECM stiffening is a prerequisite for cells overactivating FAK-SRC to upregulate SNAIL1 and acquire a mesenchymal phenotype, and 2) FAK-SRC inhibition of cell-cell contacts through the Receptor Protein Tyrosine Phosphates kappa leads to the acquisition of a full mesenchymal rather than a hybrid phenotype.
Project description:HMLER cells (Ras-transformed version of HMLE cells) were FACS sorted as two subpopulation using CD24 and ITGB4 antibodies. Cells which are negative for CD24 and ITGB4 were mesenchymal in morphology, while cells which are psotive for CD24 and ITGB4 were epithelial. We performed RNA sequencing and expression of several epithelial and mesenchymal phenotype associated proteins were consistent with their mrphology. In addition to the EMT related genes, these two population expressed some unique set of genes.
Project description:Bone marrow (BM) mesenchymal stem/stromal cells are non-hematopoietic (CD45-), non-endothelial (CD31-) multipotential cells capable to differentiate into osteoblastes, chondrocytes and adipocytes. In addition, different subpopulations of MSCs and some of their derivatives (early osteoblastic lineage cells) were shown to form HSC-niche. This makes a complex picture of the relationship between MSCs and HSCs. Despite growing data in mice model, few describe the human counterpart. The BM CD200+ and CD271+ fractions were previously shown to be enriched in native MSCs in human. Herein, we found heterogeneity in expression of CD200 within CD45-/CD31-/CD271+ human BM fraction. We thus selected CD200+ and CD200- cells from CD45-/CD31-/CD271+ BM samples and we analyzed their transcriptome. The differential display of gene expression between these two types of BM fractions will give new insights in the identification of native human MSCs.
Project description:p63 is a transcription factor central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in a human prostate cell culture model, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT by a gain and loss of function approach. Over-expression of the predominant epithelial isoform DNp63a in mesenchymal EPT1B8 cells led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses found that DNp63a induced gene modules involving cell adhesion genes in mesenchymal like cells. Genome-wide analysis of p63 binding sites by ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells.CDH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with DNp63a to reverse the mesenchymal phenotype in EPT1 cells. The partial reversion of epithelial to mesenchymal transition might reflect the ability of DNp63a, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of DNp63a expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation. Examination of p63 binding profile in prostate cell model EP156T with EPT1 as negative control.
Project description:Tumor heterogeneity and cisplatin resistance are major causes of tumor relapse and poor survival. Here, we show that in lung cancer, interaction between paxillin (PXN) and integrin b4 (ITGB4), components of the focal adhesion (FA) complex, contributes to cisplatin resistance. Knocking down PXN and ITGB4 attenuated cell growth and improved cisplatin sensitivity, both in 2D and 3D cultures. PXN and ITGB4 independently regulated expression of several genes. In addition, they also regulated expression of common genes including USP1 and VDAC1 that are required for maintaining genomic stability and mitochondrial function, respectively. Mathematical modelling suggested that bistability could lead to stochastic phenotypic switching between cisplatin-sensitive and resistant states in these cells. Consistently, purified subpopulations of sensitive and resistant cells recreated the mixed parental population when cultured separately. Altogether, these data point to an unexpected role of the FA complex in cisplatin resistance, and highlight a novel non-genetic mechanism.