Cell softness regulates tumorigenicity and stemness of cancer cells
Ontology highlight
ABSTRACT:
SUBMITTER: Prof. Bo Huang
PROVIDER: S-SCDT-EMBOJ-2020-106123 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Identifying and sorting highly tumorigenic and metastatic tumor cells from a heterogeneous cell population is a daunting challenge. Here, we show that microfluidic devices can be used to sort marker-based heterogeneous cancer stem cells (CSC) into mechanically stiff and soft subpopulations. The isolated soft tumor cells (< 400 Pa) but not the stiff ones (> 700 Pa) can form a tumor in immunocompetent mice with 100 cells per inoculation. Notably, only the soft, but not the stiff cells, isolated from CD133+ , ALDH+ , or side population CSCs, are able to form a tumor with only 100 cells in NOD-SCID or immunocompetent mice. The Wnt signaling protein BCL9L is upregulated in soft tumor cells and regulates their stemness and tumorigenicity. Clinically, BCL9L expression is correlated with a worse prognosis. Our findings suggest that the intrinsic softness is a unique marker of highly tumorigenic and metastatic tumor cells.
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Project description:BackgroundEukaryotic histone methyltransferases 2 (EHMT2 or G9A) has been regarded as a potential target for non-small cell lung cancer (NSCLC) therapy. This study investigated the regulatory roles of G9A in tumorigenesis and stemness in NSCLC. We isolated and enriched tumor-initiating cells (TIC) from surgically resected NSCLC tissues by FACS and sphere formation assays. We then knocked down G9A using shRNA and carried out genome-wide 850K methylation array and RNA sequencing analyses. We carried out in vivo tumorigenecity asssay using mice xenografts and examined G9A interactions with its novel target using chromatin Immunoprecipitation (ChIP).ResultsWe identified 67 genes hypomethylated and 143 genes upregulated following G9A knockdown of which 43 genes were both hypomethylated and upregulated. We selected six genes (CDYL2, DPP4, SP5, FOXP1, STAMBPL1, and ROBO1) for validation. In addition, G9A expression was higher in TICs and targeting G9a by shRNA knockdown or by selective inhibitor UNC0642 significantly inhibited the expression of cancer stem cell markers and sphere forming capacity, in vitro proliferation, and in vivo growth. Further, transient overexpression of FOXP1, a protein may promote normal stem cell differentiation, in TICs resulted in downregulation of stem cell markers and sphere forming capacity and cell proliferation in vitro indicating that the genes we identified are directly regulated by G9A through aberrant DNA methylation and subsequent expression. Similarly, ChIP assay has shown that G9a interacts with its target genes through H3K9me2 and downregulation of H3K9me2 following G9a knockdown disrupts its interaction with its target genes.ConclusionsThese data suggest that G9A is involved in lung cancer stemness through epigenetic mechanisms of maintaining DNA methylation of multiple lung cancer stem cell genes and their expression. Further, targeting G9A or its downstream genes could be a novel therapeutic approach in treating NSCLC patients.
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Project description:BackgroundPrevious studies demonstrated the dependence of cancer on nerve. Recently, a growing number of studies reveal that cancer cells share the property and regulatory network with neural stem/progenitor cells. However, relationship between the property of neural stemness and cell tumorigenicity is unknown.ResultsWe show that neural stem/progenitor cells, but not non-neural embryonic or somatic stem/progenitor cell types, exhibit tumorigenicity and the potential for differentiation into tissue types of all germ layers when they are placed in non-native environment by transplantation into immunodeficient nude mice. Likewise, cancer cells capable of tumor initiation have the property of neural stemness because of their abilities in neurosphere formation in neural stem cell-specific serum-free medium and in differentiation potential, in addition to their neuronal differentiation potential that was characterized previously. Moreover, loss of a pro-differentiation factor in myoblasts, which have no tumorigenicity, lead to the loss of myoblast identity, and gain of the property of neural stemness, tumorigenicity and potential for re-differentiation. By contrast, loss of neural stemness via differentiation results in the loss of tumorigenicity. These suggest that the property of neural stemness contributes to cell tumorigenicity, and tumor phenotypic heterogeneity might be an effect of differentiation potential of neural stemness. Bioinformatic analysis reveals that neural genes in general are correlated with embryonic development and cancer, in addition to their role in neural development; whereas non-neural genes are not. Most of neural specific genes emerged in typical species representing transition from unicellularity to multicellularity during evolution. Genes in Monosiga brevicollis, a unicellular species that is a closest known relative of metazoans, are biased toward neural cells.ConclusionsWe suggest that the property of neural stemness is the source of cell tumorigenicity. This is due to that neural biased unicellular state is the ground state for multicellularity and hence cell type diversification or differentiation during evolution, and tumorigenesis is a process of restoration of neural ground state in somatic cells along a default route that is pre-determined by an evolutionary advantage of neural state.
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Project description:Cancer stem cells (CSCs) are associated with cancer recurrence following radio/chemotherapy owing to their high resistance to therapeutic intervention. In this study, we investigated the role of exostoxin 1 (EXT1), an endoplasmic reticulum (ER)-residing type II transmembrane glycoprotein, in cancer cell stemness. DNA microarray analysis revealed that doxorubicin-resistant MCF7/ADR cells have high levels of EXT1 expression compared to its parental cell line, MCF7. These cells showed significantly higher populations of CSCs and larger populations of aldehyde dehydrogenase (ALDH+) and CD44+/CD24-cells, as compared to MCF7 cells. siRNA-mediated knockdown of EXT1 in MCF7/ADR cells significantly reduced cancer stem cell markers, populations of ALDH+and CD44+/CD24- cells, mRNA and protein expression for CD44, and mammosphere number. Furthermore, epithelial mesenchymal transition (EMT) markers and migratory behavior were also repressed with reduced EXT1. In an in vitro soft agar colony formation assay, EXT1 knockdown by short hairpin RNA (shRNA) reduced the colony formation ability of these cells. Based on these results, we suggest that EXT1 could be a promising novel target to overcome cancer cell stemness in anthracycline-based therapeutic resistance.
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Project description:DNA double-strand breaks (DSBs) are traditionally associated with cancer through their abilities to cause chromosomal instabilities or gene mutations. Here we report a new class of self-inflicted DNA DSBs that can drive tumor growth irrespective of their effects on genomic stability. We discover a mechanism through which cancer cells cause DSBs in their own genome spontaneously independent of reactive oxygen species or replication stress. In this mechanism, low-level cytochrome c leakage from the mitochondria leads to sublethal activation of apoptotic caspases and nucleases, which causes DNA DSBs. In response to these spontaneous DNA DSBs, ATM, a key factor involved in DNA damage response, is constitutively activated. Activated ATM leads to activation of transcription factors NF-?B and STAT3, known drivers of tumor growth. Moreover, self-inflicted DNA DSB formation and ATM activation are important in sustaining the stemness of patient-derived glioma cells. In human tumor tissues, elevated levels of activated ATM correlate with poor patient survival. Self-inflicted DNA DSBs therefore are functionally important for maintaining the malignancy of cancer cells.
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Project description:BackgroundPancreatic cancer (PC) represents one of the most aggressive forms of cancer. The role of long non-coding RNAs (lncRNAs) has been highlighted in various malignancies including PC. The aim of the present study was to investigate the effects associated with actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) on the progression of PC and the underlying mechanism.MethodsMicroarray-based gene expression profiling of PC was performed to identify PC-related lncRNAs, after which the expression of AFAP1-AS1 and cancer stem cell (CSC) markers in PC tissues and cells were determined accordingly. The potential microRNA-384 (miR-384) capable of binding to AFAP1-AS1, in addition to its ability to regulate activin receptor A type I (ACVR1) were analyzed. In order to investigate the effect of the AFAP1-AS1/miR-384/ACVR1 axis on self-renewal ability, tumorigenicity, invasion, migration and stemness of PC cells, shRNA-AFAP1-AS1, miR-384 mimic and inhibitor were cloned into cells.ResultsHigh expression of AFAP1-AS1 and ACVR1 with low expression of miR-384 were detected in PC tissues. ACVR1 was determined to be down-regulated when miR-384 was overexpressed, while the inhibition of AFAP1-AS1 decreased its ability to binding competitively to miR-384, resulting in the down-regulation of ACVR1 and enhancing miR-384 expression, ultimately inhibiting the progression of PC. The knockdown of AFAP1-AS1 or overexpression of miR-384 was confirmed to impair PC cell self-renewal ability, tumorigenicity, invasion, migration and stemness.ConclusionsTaken together, AFAP1-AS1 functions as an endogenous RNA by competitively binding to miR-384 to regulate ACVR1, thus conferring inhibitory effects on PC cell stemness and tumorigenicity.
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Project description:Neural stemness is suggested to be the ground state of tumorigenicity and pluripotent differentiation potential. However, the relationship between these cell properties is unclear. Here, by disrupting the neural regulatory network in neural stem and cancer cells and by serial transplantation of cancer cells, we show that tumorigenicity and pluripotent differentiation potential are coupled cell properties unified by neural stemness. We show that loss of neural stemness via inhibition of SETDB1, an oncoprotein with enriched expression in embryonic neural cells during vertebrate embryogenesis, led to neuronal differentiation with reduced tumorigenicity and pluripotent differentiation potential in neural stem and cancer cells, whereas enhancement of neural stemness by SETDB1 overexpression caused the opposite effects. SETDB1 maintains a regulatory network comprising proteins involved in developmental programs and basic cellular functional machineries, including epigenetic modifications (EZH2), ribosome biogenesis (RPS3), translation initiation (EIF4G), and spliceosome assembly (SF3B1); all of these proteins are enriched in embryonic neural cells and play active roles in cancers. In addition, SETDB1 represses the transcription of genes promoting differentiation and cell cycle and growth arrest. Serial transplantation of cancer cells showed that neural stemness, tumorigenicity, and pluripotent differentiation potential were simultaneously enhanced; these effects were accompanied by increased expression of proteins involved in developmental programs and basic machineries, including SETDB1 and the abovementioned proteins, as well as by increased alternative splicing events. These results indicate that basic machineries work together to define a highly proliferative state with pluripotent differentiation potential and also suggest that neural stemness unifies tumorigenicity and differentiation potential.
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Project description:BACKGROUND:Glioblastoma is devastating cancer with a high frequency of occurrence and poor survival rate and it is urgent to discover novel glioblastoma-specific antigens for the therapy. Cancer-germline genes are known to be related to the formation and progression of several cancer types by promoting tumor transformation. Dazl is one such germline gene and is up-regulated in a few germ cell cancers. In this study, we analyzed the expression of Dazl in human glioblastoma tissues and cells, and investigated its significance in proliferation, migration, invasion and chemoresistance of the glioblastoma cell lines. METHODS:We evaluated the expression of Dazl in different pathologic grades of glioblastoma tissues by immunohistochemistry. We assessed the expression of Dazl in glioblastoma cells and normal human astrocytes (NHA) cells by western blotting and RT-qPCR. Then we generated Dazl knockout glioblastoma cell lines using the CRISPR/Cas9 gene-editing technology to explore the cellular function of Dazl. We detected the proliferation and germline traits via CCK-8 assays and alkaline phosphatase staining, respectively. Boyden chamber assays were performed to measure glioblastoma cell migration and invasion. Crystal violet staining was used to determine the number of viable cells after the treatment of Doxorubicin and Temozolomide. Finally, we used subcutaneous xenograft studies to measure the growth of tumors in vivo. RESULTS:We found that Dazl was upregulated in glioblastoma tissues and glioblastoma cell lines. Dazl knockdown glioblastoma cells showed decreased cellular proliferation, migration, invasion, and resistance in vitro, and inhibited the initiation of glioblastoma in vivo. The glioblastoma cell lines A172, U251, and LN229 were found to express stem cell markers CD133, Oct4, Nanog, and Sox2. The expression of these markers was downregulated in Dazl-deficient cells. CONCLUSIONS:Our results indicated that Dazl contributes to the tumorigenicity of glioblastoma via reducing cell stemness. Therefore, cancer-germline genes might represent a new paradigm of glioblastoma-initiating cells in the treatment of malignant tumors.
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