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1.
Oncogene ; 39(9): 1957-1968, 2020 02.
Article in English | MEDLINE | ID: mdl-31772328

ABSTRACT

High YAP activity is associated with poor prognosis human breast cancers, but its role during the initial stage of mammary cell transformation is unknown. To address this question, we designed experiments that exploit the ability of KRASG12D-transduced subsets of freshly isolated normal human mammary cells to form invasive tumors rapidly and efficiently when transplanted into immunodeficient mice. Initial examination of the newly developing tumors thus generated revealed a consistent marked loss of nuclear YAP, independent of the initial primary human mammary cell type transduced. Conversely, co-transduction of the same subsets of primary human mammary cells with KRASG12D plus the constitutively active YAPS127A prevented tumor formation. These findings contrast with the enhanced display of transformed properties obtained when the immortalized, but non-tumorigenic MCF10A cells are transduced just with YAPS127A. In addition, we show that YAPS127A-transduction of the human MDA-MB-231 breast cancer cell line (that carry a similar KRAS mutation) enhances their metastatic activity in vivo. We also discover that the KRASG12D-induced early loss of YAP in primary human mammary cells is associated with their induced secretion of amphiregulin. Collectively, these findings suggest that YAP can differentially affect the acquisition of malignant properties by human mammary cells at different stages of their transformation.


Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Breast Neoplasms/pathology , Breast/pathology , Carcinogenesis/pathology , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Transcription Factors/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amphiregulin/genetics , Amphiregulin/metabolism , Animals , Apoptosis , Breast/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Proliferation , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , YAP-Signaling Proteins
2.
EMBO Rep ; 20(12): e48375, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31668005

ABSTRACT

Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5'-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.


Subject(s)
Antineoplastic Agents/therapeutic use , Benzamides/therapeutic use , Bone Neoplasms/drug therapy , Histone Deacetylase Inhibitors/therapeutic use , Pyridines/therapeutic use , Sarcoma, Ewing/drug therapy , Transcription Factors/metabolism , Acetylation , Animals , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Line, Tumor , Cells, Cultured , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , NF-E2-Related Factor 2/metabolism , Neoplasm Metastasis , Oxidative Stress , Sarcoma, Ewing/metabolism , Sarcoma, Ewing/pathology
3.
J Biol Chem ; 293(32): 12502-12515, 2018 08 10.
Article in English | MEDLINE | ID: mdl-29903916

ABSTRACT

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.


Subject(s)
Oncogene Proteins, Fusion/metabolism , Polyubiquitin/metabolism , Proteolysis , Receptors, Somatomedin/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , Cells, Cultured , Humans , Oncogene Proteins, Fusion/genetics , Phosphorylation , Receptor, IGF Type 1 , Receptors, Somatomedin/genetics , Receptors, Somatomedin/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Ubiquitination
4.
Oncogenesis ; 7(2): 14, 2018 Feb 03.
Article in English | MEDLINE | ID: mdl-29396433

ABSTRACT

Cancer cells utilize lysosomes for invasion and metastasis. Myeloid Zinc Finger1 (MZF1) is an ErbB2-responsive transcription factor that promotes invasion of breast cancer cells via upregulation of lysosomal cathepsins B and L. Here we identify let-7 microRNA, a well-known tumor suppressor in breast cancer, as a direct negative regulator of MZF1. Analysis of primary breast cancer tissues reveals a gradual upregulation of MZF1 from normal breast epithelium to invasive ductal carcinoma and a negative correlation between several let-7 family members and MZF1 mRNA, suggesting that the inverse regulatory relationship between let-7 and MZF1 may play a role in the development of invasive breast cancer. Furthermore, we show that MZF1 regulates lysosome trafficking in ErbB2-positive breast cancer cells. In line with this, MZF1 depletion or let-7 expression inhibits invasion-promoting anterograde trafficking of lysosomes and invasion of ErbB2-expressing MCF7 spheres. The results presented here link MZF1 and let-7 to lysosomal processes in ErbB2-positive breast cancer cells that in non-cancerous cells have primarily been connected to the transcription factor EB. Identifying MZF1 and let-7 as regulators of lysosome distribution in invasive breast cancer cells, uncouples cancer-associated, invasion-promoting lysosomal alterations from normal lysosomal functions and thus opens up new possibilities for the therapeutic targeting of cancer lysosomes.

5.
Cell Cycle ; 11(13): 2415-6, 2012 Jul 01.
Article in English | MEDLINE | ID: mdl-22713242
6.
Mol Cell ; 45(6): 764-76, 2012 Mar 30.
Article in English | MEDLINE | ID: mdl-22464443

ABSTRACT

Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified signaling network activates the transcription of cathepsin B gene (CTSB) via myeloid zinc finger-1 transcription factor that binds to an ErbB2-responsive enhancer element in the first intron of CTSB. This work provides a model system for ErbB2-induced breast cancer cell invasiveness, reveals a signaling network that is crucial for invasion in vitro, and defines a specific role and targets for the identified serine-threonine kinases.


Subject(s)
Breast Neoplasms/pathology , Cathepsin B/genetics , Cathepsin B/metabolism , Kruppel-Like Transcription Factors/metabolism , Receptor, ErbB-2/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cathepsin L/genetics , Cathepsin L/metabolism , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Kruppel-Like Transcription Factors/genetics , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Myotonin-Protein Kinase , Neoplasm Invasiveness , Promoter Regions, Genetic , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Protein c-ets-1/genetics , Proto-Oncogene Protein c-ets-1/metabolism , Receptor, ErbB-2/genetics , Response Elements , Signal Transduction , p21-Activated Kinases/genetics , p21-Activated Kinases/metabolism
7.
Cancer Res ; 68(16): 6623-33, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18701486

ABSTRACT

Expression and activity of lysosomal cysteine cathepsins correlate with the metastatic capacity and aggressiveness of tumors. Here, we show that transformation of murine embryonic fibroblasts with v-H-ras or c-src(Y527F) changes the distribution, density, and ultrastructure of the lysosomes, decreases the levels of lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in an extracellular signal-regulated kinase (ERK)- and cathepsin-dependent manner, and sensitizes the cells to lysosomal cell death pathways induced by various anticancer drugs (i.e., cisplatin, etoposide, doxorubicin, and siramesine). Importantly, K-ras and erbb2 elicit a similar ERK-mediated activation of cysteine cathepsins, cathepsin-dependent down-regulation of LAMPs, and increased drug sensitivity in human colon and breast carcinoma cells, respectively. Notably, reconstitution of LAMP levels by ectopic expression or by cathepsin inhibitors protects transformed cells against the lysosomal cell death pathway. Furthermore, knockdown of either lamp1 or lamp2 is sufficient to sensitize the cells to siramesine-induced cell death and photo-oxidation-induced lysosomal destabilization. Thus, the transformation-associated ERK-mediated up-regulation of cysteine cathepsin expression and activity leads to a decrease in the levels of LAMPs, which in turn contributes to the enhanced sensitivity of transformed cells to drugs that trigger lysosomal membrane permeabilization. These data indicate that aggressive cancers with high cysteine cathepsin levels are especially sensitive to lysosomal cell death pathways and encourage the further development of lysosome-targeting compounds for cancer therapy.


Subject(s)
Apoptosis/physiology , Cell Transformation, Neoplastic , Colonic Neoplasms/metabolism , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomal-Associated Membrane Protein 2/metabolism , Lysosomes/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cathepsins/metabolism , Cell Communication , Cell Membrane Permeability , Cells, Cultured , Colonic Neoplasms/pathology , Down-Regulation , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Genes, ras/physiology , Humans , Lysosomal-Associated Membrane Protein 1/genetics , Lysosomal-Associated Membrane Protein 2/genetics , Lysosomes/drug effects , Mice , NIH 3T3 Cells , Proto-Oncogene Proteins c-raf/metabolism , Proto-Oncogene Proteins p21(ras)/physiology , RNA Interference
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