AP-1 regulates heterogeneous cellular dormancy in TNBC II
Ontology highlight
ABSTRACT: Slow-cycling cells (SCCs) are a heterogeneous subpopulation of tumor cells. They pre-exist in tumor tissues and evade killing by chemotherapeutics that target mitosis due to their dormant nature. We here utilized PKH26 retaining method to label and isolate SCCs, and RNA-seq was performed to understand the gene expression characteristics of SCCs. Here we reported that up-regulations of AP-1 subunits regulate triple negative breast cancer (TNBC) dormancy by up-regulating the expression of CDKN1A and GADD45 family as transcription factors.
Project description:Slow-cycling cells (SCCs) are a heterogeneous subpopulation of tumor cells. They pre-exist in tumor tissues and evade killing by chemotherapeutics that target mitosis due to their dormant nature. We here utilized varieties of fluorescent dyes or proteins (Claret, H2BeGFP, and Violet) retaining method to label and isolate SCCs, and RNA-seq was performed to understand the gene expression characteristics of subpopulations of SCCs.
Project description:The genes whose transcription was significantly altered by metformin were investigated in H1299 cells by RNA-seq. Significantly up- or down-regulated genes were identified by cutoff value of |FC| >= 1.5. Metformin significantly downregulated mRNA levels of 499 genes while upregulating 615. KEGG (Kyoto Encyclopedia of Genes and Genomes) gene enrichment analysis revealed that the highly ranked clusters were cell cycle, apoptosis, cellular senescence and p53 signaling pathways. Multiple cell cycle checkpoint genes such as CCNA, CCNB, CCNE, CDK1, E2F2, E2F6, and E2F8 were downregulated, while certain cell cycle inhibitors such as CDKN2B (p15), CDKN1A (p21), DDIT4, and GADD45 were upregulated.
Project description:Slow-cycling/dormant cancer cells (SCCs) are thought to cause cancer relapse, but the underlying biology remains obscure. A subpopulation of SCCs has been found even in rapidly growing tumors and cancer cell lines. To investigate the mechanism underlying cellular dormancy, we obtained SCCs from three NSCLC cell lines (H460, H1299, and SK-MES-1) and PDX tumor by using a cell proliferation-dependent fluorescent dye (CSFE).
Project description:Using the immunocompetent bone-metastatic RM1 murine model of prostate cancer, the transriptomes of proliferating (PKH26-) eCFP+/luc2+ RM1 single cells (referred to as RM1536) were compared to dormant (PKH26+) RM1536 single cells dervied from bioluminescence imaging-verified bone metastases in mice at ~d16 post-intracardiac injection of PKH26 labelled RM1536 cells
Project description:We established fallopian tube epithelial organoids from human fallopian tube tissues and cultured them in the organoid culturing cocktail medium. To investigate the heterogeneity of proliferation in the fallopian tube epithelial organoids, we stained the fallopian tube epithelial cells by a fluorescent membrane dye PKH26. As culturing them, the intensity of PKH26 was reduced in accordance with the proliferative level of each cell. We sorted the organoid cells by FACS into PKH26-retained slow cycling cells and PKH26-reduced proliferative cells, and compared their transcriptomic characteristics by bulk RNA sequencing and gene set enrichment analysis (GSEA). The result of GSEA showed that several pathways related to cell cycle were downregulated in PKH26-retained cells. So this dataset is supposed to be suitable for the analysis of quiescent cells in human fallopian tube epithelium.
Project description:Doxorubicin is considered one of the most potent established chemotherapeutics in the treatment of liposarcoma; however, the response rates usually below 30%, are still disappointing. This study was performed to identify gene expression changes in liposarcoma after doxorubicin treatment. Cells of 19 primary human liposarcoma were harvested intraoperatively and brought into cell culture. Cells were incubated with doxorubicin for 24 h, RNA was isolated and differential gene expression was analysed by the microarray technique. We used microarrays to detail the global gene expression changes following doxorubicin treatment of primary liposarcoma cell cultures Experiment Overall Design: We compared untreated primary cell cultures obtained from liposarcoma treated with doxorubicin treated cultures to determine global gene expression changes by microarray analysis
Project description:Recent data suggests that colon tumors contain a subpopulation of therapy resistant quiescent cancer stem cells (qCSCs) are the source of relapse following treatment. The isolation and molecular characterisation of qCSCs may therefore lead to the identification of novel therapeutic targets for their elimination. Label retention of the lipophilic fluorescent dye PKH26, wherein dividing cells lose the label and quiescent or slow cycling cells retain the label for an extended period of time, was used to isolate qCSCs from a panel of colon cancer patient-derived organoids (PDOs). PDOs were dissociated to single cells, labelled with PKH26 and plated in Matrigel culture. After 12 days, PDOs were processed to single cells, labelled with DAPI (to exclude dead cells) and isolated by fluorescence assisted cell sorting (FACS) into two fractions of PKH26-Negative and PKH26-Positive (label retaining) cells. Functional analyses of these cellular subpopulations demonstrated that PKH26-Positive cells are self-renewing qCSCs. RNA was collected from FAC sorted PKH26-Negative and PKH26-Positive cells and analysed by RNA-sequencing. Cells were lysed in RLTplus buffer and RNA was extracted using Qiagen RNeasy Mini Kit. RNA quality was assessed prior to sequencing. Samples were processed using NuGEN’s Ovation RNA-Seq System V2 and Ultralow V2 Library System and sequenced on an Illumina HiSeq 2500 machine as 2x125nt paired-end reads. Reads were mapped to the human reference genome (assembly hg19) using the STAR aligner (version 2.4.2a). Total read counts per gene were computed using the program “featureCounts” (version 1.4.6-p2) in the “subread” package, with the gene annotation taken from Gencode (version 19).
Project description:Using the immunocompetent bone-metastatic murine model of prostate cancer, the transcriptomes of proliferating (PKH26-) eCFP+/luc2+ RM1 cells (referred to as RM1536) were compared to FACS-isolated, proliferating (PKH26-) RM1536 cells derived from matched bone and lung metastases for preliminary assessment of gene alterations that may specifically occur in bone.