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1、Activation of Multiple Proto-oncogenicTyrosine Kinases in Breast Cancer viaLoss of the PTPN12 PhosphataseTingting Sun, Nicola Aceto, Kristen L. Meerbrey,Jessica D. Kessler,1 Verna & Marrs McLean Department of Biochemistry &Molecular Biology2 Department of Molecular & Human Genetics3 Department of Pe

2、diatricsAmong breast cancers, triple-negative breast cancer (TNBC三阴性乳腺癌) is the most poorly understood and is refractory to current targeted therapies. Triple-negative breast cancer refers to any breast cancer that does not express the genes for estrogen receptor (ER), progesterone receptor (PR) or

3、HER2. (which is one of the epithelial growth factor receptor family)mitogen-activated protein kinases,MAP RESULTSUsing a genetic screen,we identify thePTPN12 tyrosine phosphatase as a tumor suppressor in TNBC. PTPN12 potently suppresses mammary epithelial cell proliferation and transformation.Notabl

4、y, PTPN12 has not been previously implicated in tumor suppression. Three independent PTPN12 shRNAs exhibited robust cellular transformation in TLM-HMECsthe degree of depletion correlated with theseverity of the phenotype (Figures 1B and 1C). In addition,restoring PTPN12 expression with an exogenous

5、PTPN12cDNA completely suppressed transformation (Figure 1D),Collectively, these data indicate that PTPN12 is a potent suppressor of transformation in mammary epithelial cellsThis result suggesting that the tyrosine-phosphatase activity of PTPN12 is required for suppressingtransformation.The authors

6、used a quantitative proteome-wide method that combinesanti-phosphotyrosine peptide immunoprecipitation, differentialpeptide labeling, and LC-MS/MS-based phosphopeptide identi-cation and quantitation (Hsu et al., 2003) to search for phospho-tyrosine-peptides whose abundance increases when PTPN12 isde

7、pleted. The rst network consists of proteins and signaling pathways known to govern proliferation and survival in human cancer, with the RTK EGFR being a central component The second network is comprised of proteins controlling the actin cytoskeleton, consistent with the role of PTPN12 in regulating

8、 cell motility andpossibly metastasis we focused our attention on the potential regulatory interaction betweenPTPN12 and the EGFR family of receptor TKsEGFR HER2 HER3 HER4 RTKs in the HER familyThese data suggest that PTPN12 may directly interact with and inhibit EGFR/HER2 signaling to suppress tran

9、sformation.We next evaluated the effects of PTPN12 suppression on known EGFR/HER2 effector pathways (RAS/MAPK and PI3K/AKT).PTPN12 depletion led to hyperactivation of ERK/RSK/S6signaling but had no effect on phosphorylation of PI3K-effectorsAKT and S6K1 (Figure 2E) Taken together, these results indi

10、cate that loss of PTPN12 function leads to hyperphosphorylation of HER2/EGFR and activation of downstreamRAS/MAPK signaling in HMECsTo determine whether HER2/EGFR is required for transformation, TLM-HMECs expressing a dox-inducible PTPN12-shRNA were depleted of EGFR or HER2 PTPN12 Suppresses Transfo

11、rmation by Inhibiting HER2/EGFR SignalingHER2/EGFR inhibitor (lapatinib).a MEK inhibitor (U0126).Loss of PTPN12 Expression Occurs More Frequently in TNBCPTPN12 was undetectable in 37% of invasive breastcancers (example images shown in Figure 4I and Figure S5C).Strikingly, loss of PTPN12 expression o

12、ccurred most frequentlyin TNBC (60.4% of TNBCs exhibit no detectable PTPN12protein) in 185 breast cancers.By examining other suppressors of HMEC transformation,we observed that the tumor suppressor REST was also lost inprimary breast cancers (example images in Figure 5A), and theexpression of REST a

13、nd PTPN12 was highly correlated REST is a transcription factor that represses neuronal genes in non-neural tissuesREST XPTPN 12 microRNAs are an emerging class of negative regulators, and we observed that the PTPN12 30 UTR has three evolutionarily conserved binding sites for miR-124PTPN12 is frequently compromised in human TNBCs, and we identify an upstream tumor-suppre