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1.
Genes Cells ; 29(4): 301-315, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38366725

RESUMO

Antiandrogens were originally developed as therapeutic agents for prostate cancer but are also expected to be effective for breast cancer. However, the role of androgen signaling in breast cancer has long been controversial due to the limited number of experimental models. Our study aimed to comprehensively investigate the efficacy of antiandrogens on breast cancer. In the present study, a total of 18 breast cancer cell lines were treated with the agonist or antagonists of the androgen receptor (AR). Among the 18 cell lines tested, only T-47D cells proliferated in an androgen-dependent manner, while the other cell lines were almost irresponsive to AR stimulation. On the other hand, treatment with AR antagonists at relatively high doses suppressed the proliferation of not only T-47D cells but also some other cell lines including AR-low/negative cells. In addition, expression of the full-length AR and constitutively active AR splice variants, AR-V7 and ARV567es, was not correlated with sensitivity to AR antagonists. These data suggest that the antiproliferative effect of AR antagonists is AR-independent in some cases. Consistently, proliferation of AR-knockout BT-549 cells was inhibited by AR antagonists. Identification of biomarkers would be necessary to determine which breast cancer patients will benefit from these drugs.


Assuntos
Neoplasias da Mama , Neoplasias da Próstata , Masculino , Humanos , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Androgênios/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Próstata/metabolismo , Células MCF-7 , Linhagem Celular Tumoral
2.
Physiol Genomics ; 56(2): 128-135, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-37955336

RESUMO

The mammary glands are dynamic tissues affected by pregnancy-related hormones during the pregnancy-lactation cycle. Collagen production and its dynamics are essential to the remodeling of the mammary glands. Alterations of the mammary microenvironment and stromal cells during the pregnancy-lactation cycle are important for understanding the physiology of the mammary glands and the development of breast tumors. In this study, we performed an evaluation of collagen dynamics in the mammary fat pad during the pregnancy-lactation cycle. Reanalysis of single-cell RNA-sequencing (scRNA-Seq) data showed the ectopic collagen expression in the immune cells and cell-cell interactions for collagens with single-cell resolution. The scRNA-Seq data showed that type I and type III collagen were produced not only by stromal fibroblasts but also by lymphoid and myeloid cell types in the pregnancy phase. Furthermore, the total cell-cell interaction score for collagen interactions was dramatically increased in the pregnancy tissue. The data presented in this study provide evidence that immune cells contribute, at least in part, to mammary collagen dynamics. Our findings suggest that immune cells, including lymphoid and myeloid cells, might be supportive members of the extracellular matrix orchestration in the pregnancy-lactation cycle of the mammary glands.NEW & NOTEWORTHY Our study evaluated mammary gland collagen dynamics during the pregnancy-lactation cycle using single-cell RNA-sequencing data. We found ectopic collagen expression in immune cells and an increase in collagen interactions during pregnancy. Type I and type III collagen were produced by lymphoid, myeloid, and stromal fibroblast cells during pregnancy. These findings suggest that immune cells, including lymphoid and myeloid cells, play a crucial role in supporting the extracellular matrix in mammary glands during pregnancy-lactation cycles.


Assuntos
Colágeno Tipo III , Colágeno , Gravidez , Feminino , Animais , Colágeno Tipo III/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Lactação/metabolismo , Hormônios/metabolismo , RNA/metabolismo , Glândulas Mamárias Animais/metabolismo
3.
Genes Cells ; 29(2): 169-177, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38158708

RESUMO

Hypoxia-inducible factor 1 (HIF1) is a transcription factor that is stabilized under hypoxia conditions via post-translational modifications. HIF1 regulates tumor malignancy and metastasis by gene transcriptions, such as Warburg effect and angiogenesis-related genes, in cancer cells. However, the HIF1 downstream genes show varied expressional patterns in different cancer types. Herein, we performed the hierarchical clustering based on the HIF1 downstream gene expression patterns using 1406 cancer cell lines crossing 30 types of cancer to understand the relationship between HIF1 downstream genes and the metastatic potential of cancer cell lines. Two types of cancers, including bone and breast cancers, were classified based on HIF1 downstream genes with significantly altered metastatic potentials. Furthermore, different HIF1 downstream gene subsets were extracted to discriminate each subtype for these cancer types. HIF1 downstream subtyping classification will help to understand the novel insight into tumor malignancy and metastasis in each cancer type.


Assuntos
Neoplasias da Mama , Fator 1 Induzível por Hipóxia , Humanos , Feminino , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Linhagem Celular , Neoplasias da Mama/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linhagem Celular Tumoral , Hipóxia Celular/fisiologia
4.
Int J Mol Sci ; 24(18)2023 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-37762164

RESUMO

We have developed a highly sensitive promoter trap vector system using transposons to generate reporter cells with high efficiency. Using an EGFP/luciferase reporter cell clone responsive to forskolin, which is thought to activate adenylate cyclase, isolated from human chronic myelogenous leukemia cell line K562, we found several compounds unexpectedly caused reporter responses. These included tyrosine kinase inhibitors such as dasatinib and cerdulatinib, which were seemingly unrelated to the forskolin-reactive pathway. To investigate whether any other clones of forskolin-responsive cells would show the same response, nine additional forskolin-responsive clones, each with a unique integration site, were generated and quantitatively evaluated by luciferase assay. The results showed that each clone represented different response patterns to the reactive compounds. Also, it became clear that each of the reactive compounds could be profiled as a unique pattern by the 10 reporter clones. When other TKIs, mainly bcr-abl inhibitors, were evaluated using a more focused set of five reporter clones, they also showed unique profiling. Among them, dasatinib and bosutinib, and imatinib and bafetinib showed homologous profiling. The tyrosine kinase inhibitors mentioned above are approved as anticancer agents, and the system could be used for similarity evaluation, efficacy prediction, etc., in the development of new anticancer agents.


Assuntos
Inibidores de Proteínas Quinases , Humanos , Dasatinibe/farmacologia , Colforsina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Mesilato de Imatinib/farmacologia
5.
Oncol Res ; 31(5): 655-666, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37547761

RESUMO

Myc belongs to a family of proto-oncogenes that encode transcription factors. The overexpression of c-Myc causes many types of cancers. Recently, we established a system for screening c-Myc inhibitors and identified antimycin A by screening the RIKEN NPDepo chemical library. The specific mechanism of promoting tumor cell metastasis by high c-Myc expression remains to be explained. In this study, we screened approximately 5,600 microbial extracts using this system and identified a broth prepared from Streptomyces sp. RK19-A0402 strongly inhibits c-Myc transcriptional activity. After purification of the hit broth, we identified compounds closely related to the aglycone of cytovaricin and had a structure similar to that of oligomycin A. Similar to oligomycin A, the hit compounds inhibited mitochondrial complex V. The mitochondria dysfunction caused by the compounds induced the production of reactive oxygen species (ROS), and the ROS activated GSK3α/ß that phosphorylated c-Myc for ubiquitination. This study provides a successful screening strategy for identifying natural products as potential c-Myc inhibitors as potential anticancer agents.


Assuntos
Proteínas Proto-Oncogênicas c-myc , Ubiquitina , Humanos , Ubiquitina/metabolismo , Espécies Reativas de Oxigênio , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Oligomicinas
6.
J Biol Chem ; 299(9): 105083, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37495110

RESUMO

c-Myc is a critical regulator of cell proliferation and growth. Elevated levels of c-Myc cause transcriptional amplification, leading to various types of cancers. Small molecules that specifically inhibit c-Myc-dependent regulation are potentially invaluable for anticancer therapy. Because c-Myc does not have enzymatic activity or targetable pockets, researchers have attempted to obtain small molecules that inhibit c-Myc cofactors, activate c-Myc repressors, or target epigenetic modifications to regulate the chromatin of c-Myc-addicted cancer without any clinical success. In this study, we screened for c-Myc inhibitors using a cell-dependent assay system in which the expression of c-Myc and its transcriptional activity can be inferred from monomeric Keima and enhanced GFP fluorescence, respectively. We identified one mitochondrial inhibitor, antimycin A, as a hit compound. The compound enhanced the c-Myc phosphorylation of threonine-58, consequently increasing the proteasome-mediated c-Myc degradation. The mechanistic analysis of antimycin A revealed that it enhanced the degradation of c-Myc protein through the activation of glycogen synthetic kinase 3 by reactive oxygen species (ROS) from damaged mitochondria. Furthermore, we found that the inhibition of cell growth by antimycin A was caused by both ROS-dependent and ROS-independent pathways. Interestingly, ROS-dependent growth inhibition occurred only in the presence of c-Myc, which may reflect the representative features of cancer cells. Consistently, the antimycin A sensitivity of cells was correlated to the endogenous c-Myc levels in various cancer cells. Overall, our study provides an effective strategy for identifying c-Myc inhibitors and proposes a novel concept for utilizing ROS inducers for cancer therapy.


Assuntos
Antimicina A , Proteólise , Proteínas Proto-Oncogênicas c-myc , Antimicina A/farmacologia , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Fosforilação , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Treonina/metabolismo , Proteólise/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Antineoplásicos/farmacologia , Células HCT116 , Células HeLa , Sobrevivência Celular/efeitos dos fármacos , Humanos
7.
Mol Cancer Res ; 21(9): 908-921, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37310848

RESUMO

Luminal breast cancer has the highest bone metastasis frequency among all breast cancer subtypes; however, its metastatic mechanism has not been elucidated because of a lack of appropriate models. We have previously developed useful bone metastatic cell lines of luminal breast cancer using MCF7 cells. In this study, we characterized bone metastatic MCF7-BM cell lines and identified c-Jun as a novel bone metastasis marker of luminal breast cancer. The protein level of c-Jun was upregulated in MCF7-BM cells compared with that in parental cells, and its deficiency resulted in the suppression of tumor cell migration, transformation, and reduced osteolytic ability. In vivo, dominant-negative c-Jun exhibited smaller bone metastatic lesions and a lower metastatic frequency. Histologic analysis revealed that c-Jun expression was heterogeneous in bone metastatic lesions, whereas c-Jun overexpression mediated a vicious cycle between MCF7-BM cells and osteoclasts by enhancing calcium-induced migration and releasing the osteoclast activator BMP5. Pharmacological inhibition of c-Jun by the Jun amino-terminal kinase (JNK) inhibitor JNK-IN-8 effectively suppressed tumorigenesis and bone metastasis in MCF7-BM cells. Furthermore, c-Jun downstream signals were specifically correlated with the clinical prognosis of patients with the luminal subtype of breast cancer. Our results illustrate the potential benefits of a therapy that targets c-Jun to prevent bone metastasis in luminal breast cancer. IMPLICATIONS: c-Jun expression mediates bone metastasis in luminal breast cancer by forming a vicious cycle in the bone microenvironment, which reveals potential strategies for subtype-specific bone metastasis therapy.


Assuntos
Neoplasias Ósseas , Neoplasias da Mama , Feminino , Humanos , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Células MCF-7 , Osteoclastos/metabolismo , Microambiente Tumoral , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo
8.
Biochem Biophys Rep ; 34: 101480, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37180755

RESUMO

Expansion of transformed cell area is regulated by the surrounding nontransformed cells. Lonidamine (LND) was recently found to regulate transformed cell area expansion through suppressing the cell motility of nontransformed cells; however, the structure-activity relationship between LND and this inhibitory activity has yet to be elucidated. We synthesized several LND derivatives and evaluated their inhibitory activity against the expansion of transformed cell area and found that the halogenation pattern on the benzene ring moiety, the carboxylic acid moiety, and the overall hydrophobicity of the molecule were correlated with inhibition activity. We also found that the localization of the tight junction protein, zonula occludens-1 (ZO-1), in nontransformed cells was significantly altered after treatment with the LND derivatives that displayed inhibitory activity. Further studies with LND derivatives and monitoring the localization of ZO-1 may help to develop more active compounds for suppressing transformed cell area expansion and lead to new anticancer treatments.

9.
Cancer Cell Int ; 23(1): 57, 2023 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-37005661

RESUMO

BACKGROUND: In vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines. METHODS: We established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein. RESULTS: As a result, NF-κB inducing kinase (NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of NIK decreased in LM05 cells. The decline in NIK ubiquitination was attributed to the translational downregulation of cIAP1. CONCLUSIONS: Our study identified a dysregulated mechanism of NIK production by the suppression of NIK post-modification and cIAP1 translation. The aberrant NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.

10.
Genes Cells ; 28(4): 277-287, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36659836

RESUMO

The homeobox family genes are often dysregulated in various cancer types. Particularly HOXB7 amplification and overexpression correlate with poor prognosis in various cancer such as gastric, pancreatic, and lung cancers. Moreover, HOXB7 is known to contribute to cancer progression by promoting epithelial to mesenchymal transition, anticancer drug resistance, and angiogenesis. In this study, we show that HOXB7 is coamplified with ERBB2 in a subset of breast cancer patients and HOXB7 expression correlates with poor prognosis in HER2-positive breast cancer patients. This clinical observation is supported by the following results-HOXB7 overexpression in an immortalized murine mammary gland epithelial cell line NMuMG induces cellular transformation in vitro, tumorigenesis, and lung metastasis through the activation of JAK-STAT signaling.


Assuntos
Neoplasias da Mama , Neoplasias Pulmonares , Glândulas Mamárias Humanas , Humanos , Camundongos , Animais , Feminino , Genes Homeobox , Transição Epitelial-Mesenquimal , Glândulas Mamárias Humanas/metabolismo , Proteínas de Homeodomínio/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
11.
Target Oncol ; 17(6): 695-707, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36201110

RESUMO

BACKGROUND: Chromosomal aberrations involving the anaplastic lymphoma kinase (ALK) gene have been observed in approximately 4% of patients with non-small cell lung cancer (NSCLC). Although these patients clinically benefit from treatment with various ALK tyrosine kinase inhibitors (ALK-TKIs), none of these can inhibit the development of resistance mutations. Considering inevitable drug resistance and the variety of available ALK-TKIs, it is necessary to predict the pattern of drug-resistance mutations to determine the optimal treatment strategy. OBJECTIVE: We aimed to establish a polymerase chain reaction (PCR)-based system to predict the development of resistance mutations against ALK-TKIs and identify therapeutic strategies using the upcoming ALK-TKIs repotrectinib (TPX-0005) and ensartinib (X-396) following recurrence on first-line alectinib treatment for ALK-positive NSCLC. METHODS: An error-prone PCR-based method for predicting drug resistance mutations was established and the half-maximal inhibitory concentration (IC50) values of the predicted ALK mutations were evaluated in a Ba/F3 cell-based assay. RESULTS: We predicted several resistance mutations against repotrectinib and ensartinib, and demonstrated that the next-generation ALK-TKI TPX-0131, was active against repotrectinib-resistant mutations and that the FLT3 inhibitor gilteritinib was active against ensartinib-resistant mutations. CONCLUSIONS: We developed a PCR-based system for predicting drug resistance mutations. When this system was applied to repotrectinib and ensartinib, the results suggested that these drugs can be used for the second-line treatment of ALK-positive NSCLC. Predicting resistance mutations against TKIs will provide useful information to aid in the development of effective therapeutic strategies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação
12.
J Biol Chem ; 298(12): 102635, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36273581

RESUMO

Cancer cells intrinsically proliferate in an autonomous manner; however, the expansion of cancer cell areas in a tissue is known to be regulated by surrounding nontransformed cells. Whether these nontransformed cells can be targeted to control the spread of cancer cells is not understood. In this study, we established a system to evaluate the cancer-inhibitory activity of surrounding nontransformed cells and screened chemical compounds that could induce this activity. Our findings revealed that lonidamine (LND) and domperidone (DPD) inhibited expansion of oncogenic foci of KRASG12D-expressing transformed cells, whereas they did not inhibit the proliferation of monocultured KRASG12D-expressing cells. Live imaging revealed that LND and DPD suppressed the movement of nontransformed cells away from the attaching cancer cells. Moreover, we determined that LND and DPD promoted stress fiber formation, and the dominant-negative mutant of a small GTPase RhoA relieved the suppression of focus expansion, suggesting that RhoA-mediated stress fiber formation is involved in the inhibition of the movement of nontransformed cells and focus expansion. In conclusion, we suggest that elucidation of the mechanism of action of LND and DPD may lead to the development of a new type of drug that could induce the anticancer activity of surrounding nontransformed cells.


Assuntos
Antineoplásicos , Domperidona , Indazóis , Neoplasias , Domperidona/farmacologia , Indazóis/farmacologia , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Camundongos , Células Epiteliais , Glândulas Mamárias Animais/citologia , Ensaios de Seleção de Medicamentos Antitumorais
13.
Dis Model Mech ; 15(6)2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35611554

RESUMO

Gene expression analysis at the single-cell level by next-generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell-cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-sequencing datasets confirmed that the two CSC-like populations existed in TNBC xenograft models and in TNBC patients. The diversity of these multiple CSC-like populations could cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.


Assuntos
Antineoplásicos , Neoplasias de Mama Triplo Negativas , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
14.
Clin Exp Metastasis ; 39(3): 467-477, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35103869

RESUMO

Most studies on breast cancer metastasis have been performed using triple-negative breast cancer cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain metastasis ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study.


Assuntos
Neoplasias da Mama , Carcinoma Hepatocelular , Neoplasias Hepáticas , Neoplasias de Mama Triplo Negativas , Animais , Neoplasias da Mama/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
15.
Nat Immunol ; 22(11): 1391-1402, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34686865

RESUMO

Epithelial cells have an ability termed 'cell competition', which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2-ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8+ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand-receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.


Assuntos
Antígenos CD/metabolismo , Apoptose , Competição entre as Células , Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Pulmonares/metabolismo , Lesões Pré-Cancerosas/metabolismo , Receptores Imunológicos/metabolismo , Animais , Antígenos CD/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cães , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células HaCaT , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Células Madin Darby de Rim Canino , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Células RAW 264.7 , Receptores Imunológicos/genética , Estresse Mecânico , Quinases Associadas a rho/metabolismo
16.
PLoS One ; 16(5): e0251240, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33989302

RESUMO

We have established a new screening system for identifying interacting proteins by combining biomolecular fluorescence complementation (BiFC) and a transposon gene trap system. This system requires creation of a bait strain that stably expresses a fusion product of part of the fluorescent monomeric Kusabira-Green (mKG) protein to a protein of interest. A PiggyBac transposon vector is then introduced into this strain, and a sequence encoding the remainder of mKG is inserted into the genome and fused randomly with endogenous genes. The binding partner can be identified by isolating cells that fluoresce when BiFC occurs. Using this system, we screened for interactors of p65 (also known as RELA), an NF-κB subunit, and isolated a number of mKG-positive clones. 5'- or 3'-RACE to produce cDNAs encoding mKG-fragment fusion genes and subsequent reconstitution assay identified PKM, HSP90AB1, ANXA2, HSPA8, and CACYBP as p65 interactors. All of these, with the exception of CACYBP, are known regulators of NF-κB. Immunoprecipitation assay confirmed endogenously expressed CACYBP and p65 formed a complex. A reporter assay revealed that CACYBP enhanced 3κB reporter activation under TNFα stimulation. This screening system therefore represents a valuable method for identifying interacting factors that have not been identified by other methods.


Assuntos
Elementos de DNA Transponíveis/genética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/fisiologia , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Anexina A2/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Fluorescência , Células HEK293 , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Ligação Proteica , Fator de Transcrição RelA/genética
17.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672831

RESUMO

Metastasis is a complex event in cancer progression and causes most deaths from cancer. Repeated transplantation of metastatic cancer cells derived from transplanted murine organs can be used to select the population of highly metastatic cancer cells; this method is called as in vivo selection. The in vivo selection method and highly metastatic cancer cell lines have contributed to reveal the molecular mechanisms of cancer metastasis. Here, we present an overview of the methodology for the in vivo selection method. Recent comparative analysis of the transplantation methods for metastasis have revealed the divergence of metastasis gene signatures. Even cancer cells that metastasize to the same organ show various metastatic cascades and gene expression patterns by changing the transplantation method for the in vivo selection. These findings suggest that the selection of metastasis models for the study of metastasis gene signatures has the potential to influence research results. The study of novel gene signatures that are identified from novel highly metastatic cell lines and patient-derived xenografts (PDXs) will be helpful for understanding the novel mechanisms of metastasis.


Assuntos
Neoplasias da Mama/patologia , Modelos Animais de Doenças , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica
18.
Int J Mol Sci ; 21(21)2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33114327

RESUMO

Bioluminescence imaging (BLI) is useful to monitor cell movement and gene expression in live animals. However, D-luciferin has a short wavelength (560 nm) which is absorbed by tissues and the use of near-infrared (NIR) luciferin analogues enable high sensitivity in vivo BLI. The AkaLumine-AkaLuc BLI system (Aka-BLI) can detect resolution at the single-cell level; however, it has a clear hepatic background signal. Here, to enable the highly sensitive detection of bioluminescence from the surrounding liver tissues, we focused on seMpai (C15H16N3O2S) which has been synthesized as a luciferin analogue and has high luminescent abilities as same as AkaLumine. We demonstrated that seMpai BLI could detect micro-signals near the liver without any background signal. The solution of seMpai was neutral; therefore, seMpai imaging did not cause any adverse effect in mice. seMpai enabled a highly sensitive in vivo BLI as compared to previous techniques. Our findings suggest that the development of a novel mutated luciferase against seMpai may enable a highly sensitive BLI at the single-cell level without any background signal. Novel seMpai BLI system can be used for in vivo imaging in the fields of life sciences and medicine.


Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Neoplasias Hepáticas/secundário , Micrometástase de Neoplasia/diagnóstico por imagem , Tiazóis/síntese química , Animais , Feminino , Neoplasias Hepáticas/diagnóstico por imagem , Medições Luminescentes , Camundongos , Estrutura Molecular , Transplante de Neoplasias , Sensibilidade e Especificidade , Tiazóis/administração & dosagem , Tiazóis/química
19.
Cancers (Basel) ; 12(7)2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640677

RESUMO

HER2 is overexpressed in 25-30% of breast cancers, and approximately 30% of HER2-positive breast cancers metastasize to the brain. Although the incidence of brain metastasis in HER2-positive breast cancer is high, previous studies have been mainly based on cell lines of the triple-negative subtype, and the molecular mechanisms of brain metastasis in HER2-positive breast cancer are unclear. In the present study, we performed intracranial injection using nine HER2-positive breast cancer cell lines to evaluate their proliferative activity in brain tissue. Our results show that UACC-893 and MDA-MB-453 cells rapidly proliferated in the brain parenchyma, while the other seven cell lines moderately or slowly proliferated. Among these nine cell lines, the proliferative activity in brain tissue was not correlated with either the HER2 level or the HER2 phosphorylation status. To extract signature genes associated with brain colonization, we conducted microarray analysis and found that these two cell lines shared 138 gene expression patterns. Moreover, some of these genes were correlated with poor prognosis in HER2-positive breast cancer patients. Our findings might be helpful for further studying brain metastasis in HER2-positive breast cancer.

20.
Sci Rep ; 10(1): 5152, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198460

RESUMO

TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA), originally identified as an adaptor protein of TRAF6, has recently been shown to be involved in innate immunity, induced by a pathogen-associated molecular pattern (PAMP). ADP-ß-D-manno-heptose, a newly identified PAMP, binds to alpha-kinase 1 (ALPK1) and activates its kinase activity to phosphorylate TIFA. Phosphorylation triggers TIFA oligomerisation and formation of a subsequent TIFA-TRAF6 oligomeric complex for ubiquitination of TRAF6, eventually leading to NF-κB activation. However, the structural basis of TIFA-dependent TRAF6 signalling, especially oligomer formation of the TIFA-TRAF6 complex remains unknown. In the present study, we determined the crystal structures of mouse TIFA and two TIFA mutants-Thr9 mutated to either Asp or Glu to mimic the phosphorylation state-to obtain the structural information for oligomer formation of the TIFA-TRAF6 complex. Crystal structures show the dimer formation of mouse TIFA to be similar to that of human TIFA, which was previously reported. This dimeric structure is consistent with the solution structure obtained from small angle X-ray scattering analysis. In addition to the structural analysis, we examined the molecular assembly of TIFA and the TIFA-TRAF6 complex by size-exclusion chromatography, and suggested a model for the TIFA-TRAF6 signalling complex.


Assuntos
Imunidade Inata/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , NF-kappa B/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitinação
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