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ASNA1 and cisplatin resistance: studies in C. elegans and in human tumor cells
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platinum based chemotherapy is widely used to treat cancer. Cisplatin (diamminedichloroplatinum) combination treatments provide cure for metastatic testicular cancer and prolong survival for patients suffering from ovarian, head and neck, bladder and non small cell lung cancer. Tumors that initially respond to treatment may eventually acquire resistance, resulting in treatment failure. Cisplatin resistant cells are crossresistant to arsenite and antimonite and these metalloids are exported from bacteria by the ars-operon.

In this thesis, we describe the human ArsA homolog, ASNA1, as a protein involved in a novel resistance mechanism to cisplatin, arsenite and antimonite. ASNA1 was downregulated by antisense and siRNA techniques in human melanoma and ovarian carcinoma cell lines. These cells displayed increased sensitivity to arsenite and the platinum based drugs cisplatin, carboplatin and oxaliplatin. In both melanoma and ovarian carcinoma, cisplatin resistant cells overexpressed ASNA1.

Blockage of ASNA1 resulted in increased apoptosis and retarded growth, complicating further characterization of ASNA1 in human cell lines. ASNA1 also promotes insulin signaling and mediates membrane insertion of tail-anchored proteins. To explore different aspects of ASNA1 function with respect to cisplatin resistance, we used the model organism C. elegans.

In the nematode C. elegans, asna-1 (rnai) treated larvae were hypersensitive to cisplatin, arsenite and antimonite. Adult asna-1 mutant worms were cisplatin sensitive and this hypersensitivity was seen even when apoptosis was blocked. Expression of human ASNA1 rescued the cisplatin hypersensitivity in asna-1 mutants, showing conservation of function. Transgene expression of mutated forms of asna-1 separated the cisplatin hypersensitivity phenotype from the insulin signaling phenotype of asna-1 mutants. Three ASNA-1 residues, His164, Cys285 and Cys288 were identified as essential for ASNA-1 promoted cisplatin resistance but not for insulin signaling. Finally, studies of the C. elegans germline revealed increased numbers of apoptotic cells in asna-1 mutants.

In conclusion, C. elegans is a suitable model organism to identify and characterize cisplatin response mechanisms. A targeted therapy against ASNA1 could sensitize cisplatin resistant cells and improve outcome for cancer patients.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2010. , 78 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1390
Keyword [en]
ASNA1, Cisplatin, C. elegans, Drug resistance, Apoptosis
National Category
Surgery Cancer and Oncology
Research subject
Surgery; Oncology; cellforskning
Identifiers
URN: urn:nbn:se:umu:diva-37661ISBN: 978-91-7459-115-6 (print)OAI: oai:DiVA.org:umu-37661DiVA: diva2:369598
Public defence
2010-12-10, Sal B, 9tr, Tandläkarhögskolan, Norrlands Universitetssjukhus, Umeå, 11:17 (Swedish)
Opponent
Supervisors
Funder
Swedish Research Council, K2008-68X-20803-01-3
Available from: 2010-11-19 Created: 2010-11-11 Last updated: 2010-11-19Bibliographically approved
List of papers
1. ASNA1, an ATPase targeting tail-anchored proteins, regulates melanoma cell growth and sensitivity to cisplatin and arsenite
Open this publication in new window or tab >>ASNA1, an ATPase targeting tail-anchored proteins, regulates melanoma cell growth and sensitivity to cisplatin and arsenite
2009 (English)In: Cancer Chemotherapy and Pharmacology, ISSN 0344-5704, E-ISSN 1432-0843, Vol. 63, no 3, 491-499 p.Article in journal (Refereed) Published
Abstract [en]

Purpose ASNA1 is homologous to E. coli ArsA, a well characterized ATPase involved in efflux of arsenite and antimonite. Cells resistant to arsenite and antimonite are cross-resistant to the chemotherapeutic drug cisplatin. ASNA1 is also an essential ATPase for the insertion of tail-anchored proteins into ER membranes and a novel regulator of insulin secretion. The aim of this study was to determine if altered ASNA1 levels influenced growth and sensitivity to arsenite and cisplatin in human melanoma cells.

Methods Cultured melanoma T289 cells were transfected with plasmids containing sense or antisense ASNA1. Cells were exposed to cisplatin, arsenite and zinc. Cell growth and chemosensitivity were evaluated by the MTT assay and apoptosis by a TUNEL assay.

Results ASNA1 expression was necessary for growth. T289 clones with decreased ASNA1 expression exhibited 51 ± 5% longer doubling times than wildtype T289 (P = 0.0091). After exposure to cisplatin, ASNA1 downregulated cells displayed a significant increase in apoptosis. The cisplatin IC50 in ASNA1 underexpressing cells was 41.7 ± 1.8% compared to wildtype (P = 0.00097) and the arsenite IC50 was 59.9 ± 3.2% of wildtype IC50 (P = 0.0067).

Conclusions Reduced ASNA1 expression is associated with significant inhibition of cell growth, increased apoptosis and increased sensitivity to cisplatin and arsenite.

Place, publisher, year, edition, pages
Springer, 2009
Keyword
ASNA1, Cisplatin, Arsenite, Melanoma, Drug resistance
National Category
Cancer and Oncology Surgery Pharmaceutical Sciences
Research subject
cellforskning; Pharmaceutical Biochemistry; Oncology
Identifiers
urn:nbn:se:umu:diva-10210 (URN)10.1007/s00280-008-0762-2 (DOI)18478230 (PubMedID)
Available from: 2008-11-18 Created: 2008-11-18 Last updated: 2017-12-14Bibliographically approved
2. Increased sensitivity to platinating agents and arsenite in human ovarian cancer by downregulation of ASNA1
Open this publication in new window or tab >>Increased sensitivity to platinating agents and arsenite in human ovarian cancer by downregulation of ASNA1
2009 (English)In: Oncology Reports, ISSN 1021-335X, E-ISSN 1791-2431, Vol. 22, no 4, 869-875 p.Article in journal (Refereed) Published
Abstract [en]

Platinating agents constitute the first line treatment for ovarian cancer but treatment failure is common because of intrinsic and acquired resistance. Cancer cells develop the RASP-phenotype (cross resistance against arsenite, antimonite and platinum) associated with decreased accumulation of cisplatin and arsenite. ASNA1 is a possible subunit of a transport system for cisplatin and arsenite due to homology to arsA, an ATPase in the E. coli ars-complex responsible for efflux of arsenite and antimonite. Eukaryotic ASNA1 is a targeting factor for membrane insertion of tail-anchored proteins involved in the secretory pathway and cellular stress responses. The purpose with this study was to evaluate if ASNA1 expression influenced cisplatin, carboplatin, oxaliplatin or arsenite sensitivity in ovarian cancer. Human ovarian cancer cell line 2008 was transfected with a sense or an antisense ASNA1 construct. ASNA1 downregulated and overexpressing clones were identified by Western blots. Cell growth and chemosensitivity was determined by the MTT assay. Down-regulated ASNA1 expression was associated with retarded growth and increased sensitivity to cisplatin, carboplatin, oxaliplatin and arsenite whereas the cisplatin resistant 2008/A overexpresses ASNA1. These observations support the hypothesis that ASNA1 is a target to overcome platinum resistance in ovarian cancer.

Place, publisher, year, edition, pages
Spandidos Publications, 2009
Keyword
ASNA1, cisplatin, carboplatin, oxaliplatin, drug resistance, ovarian cancer
National Category
Pharmaceutical Sciences Cancer and Oncology
Research subject
Surgery; Oncology
Identifiers
urn:nbn:se:umu:diva-33774 (URN)10.3892/or_00000511 (DOI)19724867 (PubMedID)
Available from: 2010-05-06 Created: 2010-05-06 Last updated: 2017-12-12Bibliographically approved
3. ASNA-1 activity modulates sensitivity to cisplatin
Open this publication in new window or tab >>ASNA-1 activity modulates sensitivity to cisplatin
2010 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 70, no 24, 10321-10328 p.Article in journal (Refereed) Published
Abstract [en]

Cancer can be cured by platinum based chemotherapy but resistance is a major cause of treatment failure. Here we present the nematode Caenorhabditis elegans as a model to study interactions between the platinum drug cisplatin and signaling pathways in vivo. Null mutations in a single gene, asna-1, makes worms hypersensitive to cisplatin. The metalloregulated ATPase ASNA-1 promotes insulin secretion and membrane insertion of tail-anchored proteins. Using structural data from ASNA-1 homologs, we identify specific ASNA-1 mutants that are sensitive to cisplatin while still able to promote insulin signaling. Mutational analysis reveals that hypersensitivity of ASNA-1 mutants to cisplatin remains in absence of CEP-1/p53 or apoptosis. Human ASNA1 can substitute for the worm gene, indicating a conserved function. Cisplatin sensitivity is not affected by decreased insulin signaling in wild type nematodes or restored insulin signaling in asna-1 mutants. These findings provide a functional insight into ASNA-1, demonstrate that C. elegans can be used to characterize cisplatin resistance mechanisms and propose that rationally designed drugs against ASNA-1 can sensitize cancer cells to cisplatin.

Place, publisher, year, edition, pages
American Association for Cancer Research, 2010
Keyword
ASNA1, cisplatin, C. elegans, Drug resistance, arsenite
National Category
Cancer and Oncology Pharmaceutical Sciences Cell Biology
Research subject
Surgery; Oncology
Identifiers
urn:nbn:se:umu:diva-37629 (URN)10.1158/0008-5472.CAN-10-1548 (DOI)000285334200033 ()20966125 (PubMedID)
Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2017-12-12Bibliographically approved
4. ­ASNA-1 influence on apoptosis in C. elegans
Open this publication in new window or tab >>­ASNA-1 influence on apoptosis in C. elegans
(English)Manuscript (preprint) (Other academic)
Keyword
ASNA1, apoptosis, C. elegans
National Category
Cell and Molecular Biology
Research subject
cellforskning
Identifiers
urn:nbn:se:umu:diva-37646 (URN)
Available from: 2010-11-11 Created: 2010-11-11 Last updated: 2010-11-19Bibliographically approved

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