RESUMO
The MET proto-oncogene-encoded receptor tyrosine kinase (MET) and AXL receptor tyrosine kinase (AXL) are independently operating receptor tyrosine kinases (RTKs) that are functionally associated with aggressive and invasive cancer cell growth. However, how MET and AXL regulate the migratory properties of cancer cells remains largely unclear. We report here that the addition of hepatocyte growth factor (HGF), the natural ligand of MET, to serum-starved human glioblastoma cells induces the rapid activation of both MET and AXL and formation of highly polarized MET-AXL clusters on the plasma membrane. HGF also promoted the formation of the MET and AXL protein complexes and phosphorylation of AXL, independent of AXL's ligand, growth arrest-specific 6 (GAS6). The HGF-induced MET-AXL complex stimulated rapid and dynamic cytoskeleton reorganization by activating the small GTPase RAC1, a process requiring both MET and AXL kinase activities. We further found that HGF also promotes the recruitment of ELMO2 and DOCK180, a bipartite guanine nucleotide exchange factor for RAC1, to the MET-AXL complex and thereby stimulates the RAC1-dependent cytoskeleton reorganization. We also demonstrated that the MET-AXL-ELMO2-DOCK180 complex is critical for HGF-induced cell migration and invasion in glioblastoma or other cancer cells. Our findings uncover a critical HGF-dependent signaling pathway that involves the assembly of a large protein complex consisting of MET, AXL, ELMO2, and DOCK180 on the plasma membrane, leading to RAC1-dependent cell migration and invasion in various cancer cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento de Hepatócito/farmacologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Invasividade Neoplásica , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Transporte Proteico/efeitos dos fármacos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/agonistas , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Receptor Tirosina Quinase AxlRESUMO
Receptor tyrosine kinases (RTKs) are embedded in the lipid bilayer of the plasma membrane, but the specific roles of various lipids in cell signaling remain largely uncharacterized. We have previously found that acid sphingomyelinase (ASM; also known as SMPD1) regulates the conserved DAF-2 (the ortholog IGF-1R in mammals) RTK signaling pathway in Caenorhabditis elegans How ASM and its catalytic products, ceramides, control RTK signaling pathways remain unclear. Here, we report that ASM regulates the homeostasis of Met, an RTK that is frequently overexpressed in various cancers. Inactivation of ASM led to a rapid loss of Met from the plasma membrane, reduced Met phosphorylation and activation, and induced Met accumulation in the trans-Golgi network (TGN). However, trafficking of integrin ß3 and vesicular stomatitis virus glycoprotein (VSVG) was largely unaffected. Knockdown of syntaxin 6 (STX6) also blocked the Golgi exit of Met. Depletion of either ASM or STX6 led to aberrant trafficking of Met to lysosomes, promoting its degradation. Our studies reveal that ASM and ceramides, together with STX6 and cholesterol, constitute a new regulatory mechanism for the exit of Met from the Golgi during its biosynthetic route, which is used to rapidly replenish and regulate the plasma membrane levels of Met in various cancer cells.
Assuntos
Membrana Celular/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Colesterol/metabolismo , Ativação Enzimática , Feminino , Humanos , Integrina beta3/metabolismo , Ligantes , Melanoma/metabolismo , Melanoma/patologia , Glicoproteínas de Membrana/metabolismo , Neoplasias/patologia , Fosfotirosina/metabolismo , Proteínas Qa-SNARE/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Proteínas do Envelope Viral/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher levels of RS in RBCC, compared to the parental cells. The mechanisms by which CHK1 inhibition led to specific increase of RS in RBCC were related to the interruptions in the replication fork dynamics and the homologous recombination (HR). In summary, RBCC activate oncogenic pathways and thus depend upon mechanisms controlled by CHK1 signaling to maintain RS under control for survival. Our study provided the first example where upregulating RS by CHK1 inhibitor contributes to the specific killing of RBCC, and highlight the importance of the CHK1 as a potential target for treatment of radioresistant cancer cells.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reparo do DNA/genética , Recombinação Homóloga/genética , Humanos , Células MCF-7 , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Tiofenos/farmacologia , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Exposure to genotoxic agents, such as ionizing radiation (IR), produces DNA damage, leading to DNA double-strand breaks (DSB); IR toxicity is augmented when the DNA repair is impaired. We reported that radiosensitization by a PARP inhibitor (PARPi) was highly prominent in prostate cancer cells expressing the TMPRSS2-ERG gene fusion protein. Here, we show that TMPRSS2-ERG blocks nonhomologous end-joining (NHEJ) DNA repair by inhibiting DNA-PKcs. VCaP cells, which harbor TMPRSS2-ERG and PC3 cells that stably express it, displayed γH2AX and 53BP1 foci constitutively, indicating persistent DNA damage that was absent if TMPRSS2-ERG was depleted by siRNA in VCaP cells. The extent of DNA damage was enhanced and associated with TMPRSS2-ERG's ability to inhibit DNA-PKcs function, as indicated by its own phosphorylation (Thr2609, Ser2056) and that of its substrate, Ser1778-53BP1. DNA-PKcs deficiency caused by TMPRSS2-ERG destabilized critical NHEJ components on chromatin. Thus, XRCC4 was not recruited to chromatin, with retention of other NHEJ core factors being reduced. DNA-PKcs autophosphorylation was restored to the level of parental cells when TMPRSS2-ERG was depleted by siRNA. Following IR, TMPRSS2-ERG-expressing PC3 cells had elevated Rad51 foci and homologous recombination (HR) activity, indicating that HR compensated for defective NHEJ in these cells, hence addressing why TMPRSS2-ERG alone did not lead to radiosensitization. However, the presence of TMPRSS2-ERG, by inhibiting NHEJ DNA repair, enhanced PARPi-mediated radiosensitization. IR in combination with PARPi resulted in enhanced DNA damage in TMPRSS2-ERG-expressing cells. Therefore, by inhibiting NHEJ, TMPRSS2-ERG provides a synthetic lethal interaction with PARPi in prostate cancer patients expressing TMPRSS2-ERG.
Assuntos
Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão Oncogênica/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Proteínas Adaptadoras de Transdução de Sinal , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , Dano ao DNA , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies.
Assuntos
Alquilantes/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Regulação para Baixo/genética , Exonucleases/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Histonas/genética , Humanos , TemozolomidaRESUMO
Alternative non-homologous end joining (alt-NHEJ) was originally identified as a backup repair mechanism in the absence of classical NHEJ (c-NHEJ) factors but recent studies have demonstrated that alt-NHEJ is active even when c-NHEJ as well as homologous recombination is available. The functions of 53BP1 in NHEJ processes are not well understood. Here, we report that 53BP1 promotes DNA double-strand break (DSB) repair and genomic stability not only in c-NHEJ-proficient but also -deficient human G1-phase cells. Using an array of repair substrates we show that these effects of 53BP1 are correlated with a promotion of microhomology-mediated end-joining (MMEJ), a subtype of alt-NHEJ, in G1-phase. Consistent with a specific role in MMEJ we confirm that 53BP1 status does not affect c-NHEJ. 53BP1 supports sequence deletion during MMEJ consistent with a putative role in facilitating end-resection. Interestingly, promotion of MMEJ by 53BP1 in G1-phase cells is only observed in the presence of functional BRCA1. Depletion of both 53BP1 and BRCA1 increases repair needing microhomology usage and augments loss of DNA sequence, suggesting that MMEJ is a highly regulated DSB repair process. Together, these findings significantly expand our understanding of the cell-cycle-dependent roles of 53BP1 in DSB repair.
Assuntos
Reparo do DNA por Junção de Extremidades , Fase G1 , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Sequência de Bases , Western Blotting , Linhagem Celular , Ensaio Cometa , Primers do DNA , Imunofluorescência , Instabilidade Genômica , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Bone remodeling is tightly controlled by the actions of osteoblast and osteoclast. Impaired osteoblast proliferation and differentiation may disrupt the balance and lead to pathological symptom such as osteoporosis. To help understand the molecular mechanism of osteoblast proliferation, we performed a phenotype-driven high throughput screening with a random siRNA library, in search of novel genes that can accelerate murine preosteoblast MC3T3-E1 cell proliferation. Three siRNAs screened from the library were able to enhance MC3T3-E1 cell proliferation significantly. One of the proliferation-enhancing siRNAs (B7) was further subjected to expression profiling to pinpoint genes that putatively act down stream of it. A number of genes were regulated in response to proliferation-enhancing siRNA B7. Among these genes, Tmed2, which has never been reported yet in cell proliferation, was verified to be able to enhance MC3T3-E1 cell proliferation when over-expressed. Our screening process with random siRNA library provided an alternative strategy in addition to gene-specific siRNA library, in search of novel functional genes in genome scale.