RESUMO
Although imatinib has therapeutic activity for certain subsets of patients with mastocytosis, it is not always curative. Here, molecular mechanisms that confer imatinib resistance to neoplastic mast cells were investigated using an imatinib-sensitive canine neoplastic mast cell line VI-MC carrying a KIT c.1523A>T activating mutation. Two imatinib-resistant sublines were established by culturing VI-MC cells in increasing concentrations of imatinib (1 µM resistant, rVI-MC1; 10 µM resistant, rVI-MC10). Both sublines had a second KIT mutation c.2443G>C. Recombinant KIT with the second mutation was insensitive to 1 µM but sensitive to 10 µM imatinib. The effect of imatinib on the phosphorylation of KIT and its downstream signalling proteins was then examined using these sublines. KIT and ERK were constitutively phosphorylated in both sublines, and their phosphorylation was suppressed by 10 µM imatinib in rVI-MC1 cells. However, KIT but not ERK phosphorylation was suppressed in rVI-MC10 cells. The phosphorylation of ERK in rVI-MC10 cells was also not diminished by the Src family kinase (SFK) inhibitor dasatinib. This second mutation in KIT may play an important role in imatinib resistance in neoplastic mast cells. Furthermore, KIT/SFK-independent activation of ERK would be involved in imatinib resistance when the neoplastic cells are exposed to higher concentrations of imatinib.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Mesilato de Imatinib/farmacologia , Mastocitose/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise Mutacional de DNA , Modelos Animais de Doenças , Cães , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mastocitose/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by retreatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.
Assuntos
Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mesilato de Imatinib/administração & dosagem , Mastócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Linhagem Celular Tumoral , Cães , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/administração & dosagem , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Tumores do Estroma Gastrointestinal/veterinária , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib/efeitos adversos , Mastócitos/patologia , Mutação , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Ubiquitinação/efeitos dos fármacosRESUMO
Canine histiocytic sarcoma (HS) is an aggressive and fatal neoplasm that has a high recurrence rate and metastatic nature. In the present report, compounds were screened for their growth inhibitory activity in two HS cell lines using a chemical library known to target specific signalling pathways. Among 171 compounds screened, dasatinib, which targets several types of kinases, clearly inhibited cell growth in one of the two HS lines. The growth inhibitory properties of dasatinib were then examined using six HS cell lines and MDCK cells. Dasatinib demonstrated potent growth inhibitory activity against four HS cell lines with calculated IC50 values of 5.4-54.5nM, while the IC50 values in the other cell lines were in the micromolar range. In conclusion, a kinase enzyme targeted by dasatinib appears to be crucial for growth in some subsets of HS and the on-target activity of dasatinib could underlie the marked growth inhibition in HS cells.
Assuntos
Antineoplásicos/uso terapêutico , Doenças do Cão/tratamento farmacológico , Sarcoma Histiocítico/tratamento farmacológico , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dasatinibe , Cães , Pirimidinas/farmacologia , Tiazóis/farmacologiaRESUMO
A 10-year-old female Miniature Dachshund with a non-resectable gastrointestinal stromal tumour was treated with imatinib. The neoplastic cells had a deletion mutation (c.1667_1672del) within exon 11 of the c-kit gene, which resulted in deletion of three amino acids and insertion of one amino acid (p.Trp556_Val558delinsPhe) in the juxtamembrane domain of KIT. Following treatment with imatinib, the dog achieved partial remission on Day 21 with a continuous decrease in tumour size until Day 67 of treatment. Although no additional decrease in size was observed after Day 67 of treatment, the tumour remained stable in size as of Day 140 of treatment. The c-kit mutation found in the tumour cells appears to be a mutation driving oncogenesis, as evidenced by the partial remission elicited by imatinib in this dog.
Assuntos
Antineoplásicos/uso terapêutico , Sequência de Bases , Benzamidas/uso terapêutico , Doenças do Cão/tratamento farmacológico , Tumores do Estroma Gastrointestinal/veterinária , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/uso terapêutico , Deleção de Sequência , Animais , Doenças do Cão/genética , Doenças do Cão/patologia , Cães , Éxons , Feminino , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Mesilato de Imatinib , Reação em Cadeia da Polimerase/veterinária , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
Canine malignant melanomas are highly aggressive and fatal neoplasms. In the present report, 21 drugs that target specific signalling pathways were screened for their growth inhibitory activity on three canine malignant melanoma cell lines. The proteasome inhibitor bortezomib inhibited the growth of these cell lines. The growth inhibitory properties of bortezomib were then examined using nine canine malignant melanoma cell lines. Bortezomib demonstrated potent growth inhibitory activity in all cell lines with calculated IC50 values of 3.5-5.6 nM. Because suppression of the NF-κB pathway by preventing proteasomic degradation of I κB is an important mechanism of the anti-tumour activity of bortezomib, the activation status of and the effect of bortezomib on the NF-κB pathway were examined using a canine malignant melanoma cell line, CMM-1. The NF-κB pathway was constitutively activated in CMM-1 cells and bortezomib efficiently suppressed this activated pathway. Using a CMM-1 xenograft mouse model, bortezomib also significantly inhibited tumour growth via suppression of tumour cell proliferation. Collectively, these findings suggest that bortezomib has growth inhibitory activity against canine malignant melanomas potentially through suppression of the constitutively activated NF-κB pathway. Targeted therapy using bortezomib could therefore be beneficial in the management of canine malignant melanomas.
Assuntos
Ácidos Borônicos/farmacologia , Doenças do Cão/tratamento farmacológico , Melanoma/veterinária , NF-kappa B/metabolismo , Inibidores de Proteassoma/farmacologia , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doenças do Cão/etiologia , Cães , Melanoma/tratamento farmacológico , Melanoma/etiologia , NF-kappa B/antagonistas & inibidores , Neoplasias Cutâneas , Melanoma Maligno CutâneoRESUMO
A canine intestinal mast cell tumor with splenic metastasis was treated with imatinib. The intestinal and metastatic tumor masses markedly decreased following treatment although the clinical response was short lasting. A c-kit internal tandem duplication mutation, c.1250_1261dup, which causes an insertion of four amino acids in KIT, was identified in cDNA isolated from the tumor cells. The phosphorylation status of the mutant KIT and the effect of imatinib on its phosphorylation were examined using 293 cells transfected with c-kit carrying the c.1250_1261dup mutation. This mutation caused ligand-independent phosphorylation of KIT, which was suppressed by imatinib. Inhibition of constitutively activated mutant KIT with imatinib could underlie the tumor response in this dog.
Assuntos
Antineoplásicos/uso terapêutico , Doenças do Cão/tratamento farmacológico , Neoplasias Intestinais/veterinária , Mastocitose Sistêmica/veterinária , Piperazinas/uso terapêutico , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/uso terapêutico , Animais , Benzamidas , Doenças do Cão/genética , Doenças do Cão/imunologia , Cães , Feminino , Mesilato de Imatinib , Neoplasias Intestinais/tratamento farmacológico , Neoplasias Intestinais/genética , Neoplasias Intestinais/imunologia , Mastocitose Sistêmica/tratamento farmacológico , Mastocitose Sistêmica/genética , Mastocitose Sistêmica/imunologia , Fosforilação , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Resultado do TratamentoRESUMO
The stratum corneum of epidermis is an essential barrier against the external environment and water loss. This study aimed to develop an organotypic culture model that targets the reconstruction of the stratum corneum using canine keratinocyte-derived CPEK cells. The CPEK cells cultured at the air-liquid interface became stratified and formed a stratum corneum-like layer on stratum spinosum- and stratum granulosum-like layers. The CPEK cells in the stratum granulosum-like layer expressed the cornified cell envelope (CCE)-related proteins loricrin and keratinocyte differentiation-associated protein. Organotypically cultured CPEK cells were considered to form a CCE at the stratum granulosum-like layer, allowing the formation of a stratum corneum-like layer. The organotypic culture of CPEK cells could be useful for studying the barrier function of canine stratum corneum.
Assuntos
Epiderme/metabolismo , Queratinócitos/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Diferenciação Celular , Células Cultivadas , Cães , Células Epidérmicas , Queratinócitos/citologia , Queratinas/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Target therapy using the tyrosine kinase inhibitor imatinib is one of the new therapeutic approaches for canine mast cell tumors (MCTs). In the present report, we demonstrate a clinical response to imatinib in a dog with MCT carrying a c-kit c.1523A>T mutation. Moreover, the effect of this mutation on the phosphorylation status of KIT and the inhibitory potency of imatinib on the phosphorylation of the mutant KIT were examined in vitro. A dog with a MCT tumor mass on the right forelimb sole with lymph node metastasis and mastocytemia was treated with imatinib. The MCT mass markedly shrank and mastocytemia became undetectable with 2 weeks of treatment. The lymph node enlarged by metastasis became normal in size with 5 weeks of treatment. From the sequencing analysis of c-kit in tumor cells, a substitution mutation c.1523A>T that alters the amino acid composition (p.Asn508Ile) within the extracellular domain of KIT was identified. The mutant KIT expressed on 293 cells showed ligand-independent phosphorylation and imatinib suppressed this phosphorylation in a dose-dependent manner. From these findings, imatinib was considered to elicit a clinical response in a canine case of MCT via inhibition of the constitutively activated KIT caused by a c-kit c.1523A>T mutation.