RESUMO
The detachment of normal epithelial cells from matrix triggers an apoptotic response known as anoikis, during homeostatic turnover. Metastatic tumor cells evade anoikis, by mechanisms that are only partly characterized. In particular, the epithelial-mesenchymal transition (EMT) in a subset of invasive tumor cells confers anoikis-resistance. In some cases, EMT up-regulates the cancer stem cell marker CD44S and the enzyme hyaluronic acid synthase-2 (HAS2). CD44S is the major receptor for hyaluronan in the extracellular matrix. Herein, we demonstrate that CD44S, unlike the CD44E isoform expressed in normal epithelial cells, contributes to the protection against anoikis. This protection requires the interaction of CD44S with hyaluronan (HA). CD44S-HA interaction is proposed to play an important role in tumor metastasis through enhanced cell survival under detached conditions.
Assuntos
Anoikis/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Glucuronosiltransferase/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Glucuronosiltransferase/genética , Humanos , Receptores de Hialuronatos/genética , Hialuronan Sintases , Ligação Proteica , Transdução de SinaisRESUMO
KAP1 (TRIM28) is a transcriptional regulator in embryonic development that controls stem cell self-renewal, chromatin organization, and the DNA damage response, acting as an essential corepressor for KRAB family zinc finger proteins (KRAB-ZNF). To gain insight into the function of this large gene family, we developed an antibody that recognizes the conserved zinc fingers linker region (ZnFL) in multiple KRAB-ZNF. Here, we report that the expression of many KRAB-ZNF along with active SUMOlyated KAP1 is elevated widely in human breast cancers. KAP1 silencing in breast cancer cells reduced proliferation and inhibited the growth and metastasis of tumor xenografts. Conversely, KAP1 overexpression stimulated cell proliferation and tumor growth. In cells where KAP1 was silenced, we identified multiple downregulated genes linked to tumor progression and metastasis, including EREG/epiregulin, PTGS2/COX2, MMP1, MMP2, and CD44, along with downregulation of multiple KRAB-ZNF proteins. KAP1-dependent stabilization of KRAB-ZNF required direct interactions with KAP1. Together, our results show that KAP1-mediated stimulation of multiple KRAB-ZNF contributes to the growth and metastasis of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Repressoras/biossíntese , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Neoplasias da Mama/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Galinhas , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Metástase Neoplásica , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Proteínas Repressoras/metabolismo , Sumoilação , Proteína 28 com Motivo Tripartido , Regulação para Cima , Dedos de ZincoRESUMO
O(6)-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O(6)-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O(6)-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the â¼ 5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.
