ANALYSIS OF POTENTIAL MECHANISMS INVOLVED WITH NUCLEAR XIAP-ASSOCIATED BREAST CANCER CELL GROWTH AND CHEMORESISTANCE

Vol 1, 2020 - 131335
DR - Doctoral Student
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Abstract

INTRODUCTION AND OBJECTIVE: Evasion from apoptosis is related with the drug resistance phenotype in many tumor types. X-linked inhibitor of apoptosis protein (XIAP) is an antiapoptotic protein mainly found at the cytoplasm. However, recent evidence points to a role of nuclear XIAP in poor prognosis in breast cancer. Data from our group demonstrated that nuclear XIAP conferred unfavorable clinical outcome to breast cancer patients as well as increased in vitro proliferative capacity and drug resistance in a RING domain-associated manner. Here, we aimed to understand the mechanisms involved with nuclear XIAP-associated aggressive features in breast cancer. MATERIAL AND METHOD: MCF-7 human breast cancer-derived cells were transfected with XIAP variants: pEBB (empty vector), XIAPwild type, XIAPH467A (lack of ubiquitin ligase activity), XIAPΔRING (RING deletion) and XIAPNLS C-term (Insertion of a Nuclear Localization Signal at the C-terminal end). MCF-7 cells stably overexpressing XIAP variants were generated by doxycycline-inducible Tet-On system. XIAP activity on NF-kB promoter was investigated by the luciferase assay. The modulation of nuclear import via karyoferrin β1 was assessed by INI-43 pharmacological inhibitor. The expression and localization of XIAP and potential interaction partners were investigated by subcellular fractionation, Western blotting and immunofluorescence (confocal analysis). RESULTS AND CONCLUSION: Our results show that expression of Survivin, c-IAP1 and c-Myc, proteins known to interact with XIAP, was not increased in XIAPNLS C-term overexpressing cells. Although c-Myc global expression remained unaffected, XIAP overexpression at both nucleus and cytoplasm led to nuclear exclusion of c-Myc in a RING-independent manner. We also found that p50, but not p65 subunit of NF-kB, had expression increased in XIAPNLS C-term overexpressing cells. Remarkably, NF-kB luciferase activity was significantly reduced following transfections with XIAPH467A and XIAPΔRING vectors, an effect partially reversed by transfection with XIAPNLS C-term. Interestingly, the pattern of ubiquitination in K63, but not K48 ubiquitin chains, was increased following overexpression of XIAPNLS C-term, pointing to nuclear signaling transduction. Preliminary results revealed that INI-43 treatment increases XIAP levels, indicating that inhibition of karyoferrin β1 might modulate XIAP stability in our model. In conclusion, our data suggests that the oncogenic effects mediated by nuclear XIAP are not associated with Survivin, cIAP-1 and c-Myc expression, but might involve the NF-kB pathway and trigger signaling pathways instead of proteasomal degradation of target proteins. Experiments involving the analysis of transcriptomic changes induced by XIAP variants by RNA microarray and validation of XIAP stable transfectants are currently being performed and will enable a better understanding of nuclear XIAP-mediated mechanisms in breast cancer growth and chemoresistance.

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Author

Bruna Mendonça

Oi, Thales! Agradeço pela mensagem e pela observação. Exato, a densitometria foi realizada pelo smear das marcações entre 80 e 250 KDa.  Como a XIAP já foi descrita em ubiquitinar alvos celulares por essas duas cadeias de poli-ubiquitina, nós olhamos o padrão global de marcação delas nas diferentes formas de XIAP. Realizamos replicatas desse experimentos e observamos o mesmo padrão de aumento de K63 em NLS, quando comparado aos outros mutantes, o que sugere uma relevância biológica. Para todas as replicatas, a normalização foi feita em relação ao constitutivo β-actina. A média dos valores de normalização do smear de cada mutante está representado abaixo dos blots.

 
 

Thales C. Nepomuceno

Entendi. Você acredita que avaliar essa ubiquitinação em extratos nucleares pode gerar um resultado mais acentuado?

Depois que me dei conta que hoje não era seu dia de postar. Desculpa pela msg no dia errado.

Author

Bruna Mendonça

Oi, Thales! Sem problemas. Estamos aqui para construir conhecimento! =)
Acreditamos que o resultado poderia ser mais promissor, sim. Já tentamos avaliar nos extratos com frações citoplasmáticas e nucleares, mas não conseguimos marcações com ambos anticorpos. Chegamos a aumentar a quantidade de proteína e também a concentração dos anticorpos, mas não conseguimos boas marcações. Geralmente, os extratos fracionados são mais escassos do que o lisado total. Pode ser essa uma limitação técnica.

Author

Bruna Mendonça

Oi, Thales! Sem problemas. Estamos aqui para construir conhecimento! =)
Acreditamos que o resultado poderia ser mais promissor, sim. Já tentamos avaliar nos extratos com frações citoplasmáticas e nucleares, mas não conseguimos marcações com ambos anticorpos. Chegamos a aumentar a quantidade de proteína e também a concentração dos anticorpos, mas não conseguimos boas marcações. Geralmente, os extratos fracionados são mais escassos do que o lisado total. Pode ser essa uma limitação técnica.

Thales C. Nepomuceno

Entendi. Boa sorte com os experimentos. Parabéns, mais uma vez.

Author

Bruna Mendonça

Agradeço mais uma vez! 

 

Institutions
  • 1 Laboratório de Hemato-Oncologia Celular e Molecular / Programa de Hemato-Oncologia Molecular / Instituto Nacional de Câncer (INCA), RJ, Brazil.
  • 2 Programa de Imunologia e Biologia Tumoral / Instituto Nacional de Câncer (INCA), RJ, Brazil.
  • 3 Departamento de Genética e Evolução / Universidade Federal de São Carlos (UFSCar), SP, Brazil.
Track
  • Molecular Biology
Keywords
Breast cancer
XIAP subcellular localization
Molecular mechanisms
cell growth
DRUG RESISTANCE