Project description:p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in non-transformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-?B subunit. Moreover, it partly shifts p53âs conformation and transcriptional output towards a state resembling cancer-associated p53 mutants, and endow p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently downregulated in breast cancer; we propose that such downregulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator. MCF10A cells transfected with siRNA against LATS1/2 alone, p53 alone or LATS1/2 and p53 together. Two independent MCF10A batches provided biological replicates
Project description:Linking clinical multi-omics analyses with mechanistic studies may improve the understanding of rare cancers. We leveraged two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibited outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing. This resulted in the expression of ALK variants, i.e. short transcripts (ST), which we named ALK-ST1 (consisting of exons 1-2:18–29), ALK-ST2 (1:18–29), ALK-ST3 (18–29), and ALK-ST4 (1–5:12–17). To systematically investigate the oncogenic capacity of these ALK variants, we stably expressed them in p53-deficient MCF10A human mammary epithelial cells and performed different transformation assays. These experiments demonstrated that ALK-ST1, ALK-ST2, and ALK-ST3 are oncogenic variants, while ALK-ST4 could not transform MCF10A cells. We confirmed protein expression of ALK-ST1, ALK-ST2, and ALK-ST3 by western blotting, which was not possible for ALK-ST4 due to lack of a specific antibody that binds to the N-terminus of ALK that is lost in ALK-ST4. We therefore performed mass spectrometry-based label-free quantitative proteomics on lysates from MCF10A cells stably expressing empty vector (EV), wildtype ALK (ALK-WT), or ALK-ST4 to confirm its expression.
Project description:We performed ChIP-seq analysis of p53-null HCT116 cells stably expressed wild-type p53 or mutant p53 followed by ATO treatment (PANDAs) to identify p53 binding characteristics by wild-type p53 and PANDAs.
Project description:This study compares gene expression change upon expression of Yes-associated protein (YAP) wild-type or mutants in order to establish the importance of TEAD binding and WW domains in the gene-induction function of YAP. The results indicate that gene-induction is seriously comprised in YAP-S94A (TEAD binding domain mutant) expressing cells. And mutantion of WW domains (YAP-W1W2) also affect a fraction of YAP induced genes. Therefore both TEAD binding domain and WW domains are required for the full function of YAP in gene-induction. Experiment Overall Design: Four samples are included: 1. pQCXIH vector control; 2 YAP-WT expression; 3. YAP-S94A expression; 4. YAP-W1W2 expression. Gene expression profiles of YAP wild-type or mutants expressing cells were compared to that of vector control. Experiments were done in MCF10A mammary epithelial cells.
Project description:Mammary epithelial cells MCF10A and HER2 overexpressing MCF10A cells were grown on matrigel in the absence or presence of epidermal growth factor. Cells were lysed and RNA was collected at 1.5,3,5,7,9 days.
Project description:Total RNA was extracted from five million wild type (WT) and p53 knockout (p53_KO) MCF10A cells using Qiagen RNAeasy kit. Ribosomal RNAs were removed and the rest of RNAs were subject to RNAseq.
Project description:Results of growing MCF10A cells continuously in serum free media supplemented with EGF (MCF10A) or AREG (MCF10A+AREG) followed by 24 hours of ligand withdrawl and measuring gene expression provides information as to what genes are regulated by AREG and EGF in a normal mammary epithelial cell model
Project description:The first bona fide PTP proto-oncogene was the Src-homology 2 domain-containing phosphatase SHP2 (encoded by PTPN11), an ubiquitously expressed PTP that transduces mitogenic, pro-survival, cell fate and/or pro-migratory signals from numerous growth factor-, cytokine- and extracellular matrix receptors. In malignancies, SHP2 is hyperactivated either downstream of oncoproteins or by mutations.We provide analysis of the mammary epithelial cells MCF10A overexpressing human HER2 and HER3 and grown in 3D cultures for 15 days in the presence or absence of SHP2. The human mammary epithelial cells MCF10A were transduced with a doxycycline-inducible lentiviral vector expressing a CTRL miR or SHP2 miR1 or SHP2 miR2. Cells from each group were grown in 3D cultures, and the knockdown of SHP2 was induced for 15 days. RNA was extracted for gene expression analysis.