J Gynecol Oncol.  2017 May;28(3):e33. 10.3802/jgo.2017.28.e33.

Inhibition of autophagy protein LC3A as a therapeutic target in ovarian clear cell carcinomas

• Miyamoto M
• Takano M
• Aoyama T
• Soyama H
• Yoshikawa
• Tsuda H
• Furuya K

• KMID: 2413444
• DOI: http://doi.org/10.3802/jgo.2017.28.e33

Abstract

OBJECTIVE
Ovarian clear cell carcinoma (CCC) is one of histological subtypes showing poor prognosis due to chemoresistance. The association of autophagy-related proteins and clinical implementation in CCC has not been determined.
METHODS
The present study investigated whether expression of autophagy-related protein, light chain 3A (LC3A), was related with prognoses in the patients with CCC using immuno-histochemical stainings, and whether inhibition of autophagy modified the sensitivity to cisplatin in CCC cells in vitro.
RESULTS
High expression of autophagy-related protein, LC3A, was detected in 78 cases (78%) in all CCC cases. The patients with high LC3A expression showed significantly lower response rate to primary chemotherapy (17% vs. 100%, p<0.010), and had worse progression-free survival (PFS) and overall survival (OS) compared with those with LC3A low expression. Furthermore, multivariate analyses revealed that high expression of LC3A was identified as independent worse prognostic factors for PFS and OS. Inhibition of autophagy protein LC3A using hydroxychloroquine (HCQ) increased sensitivity to cisplatin in CCC cells in vitro.
CONCLUSION
High expression of LC3A proteins was associated with lower response to platinum therapy, leading to worse prognoses in CCC. Although further studies are needed to confirm the results, inhibition of autophagy by HCQ was associated with platinum sensitivity. Autophagy protein LC3A could be a promising target for treatment for CCC.

Keyword

Ovarian Neoplasms; Adenocarcinoma, Clear Cell; Autophagy; Prognosis

MeSH Terms

Adenocarcinoma, Clear Cell/drug therapy/*metabolism/mortality/pathology
Adult
Aged
Antineoplastic Agents/*therapeutic use
Autophagy
Autophagy-Related Proteins/*metabolism
Cell Line, Tumor
Cisplatin/*therapeutic use
Drug Resistance, Neoplasm/drug effects
Enzyme Inhibitors/pharmacology
Female
Humans
Hydroxychloroquine/pharmacology
Microtubule-Associated Proteins/antagonists & inhibitors/*metabolism
Middle Aged
Ovarian Neoplasms/drug therapy/*metabolism/mortality/pathology
Prognosis
Retrospective Studies
Antineoplastic Agents
Autophagy-Related Proteins
Enzyme Inhibitors
Microtubule-Associated Proteins
Hydroxychloroquine
Cisplatin

Figure

• 

  Fig. 1. Representative IHC stains of LC3A in tissue microarray-based samples of ovarian CCCs (×10). (A) NC, (B) score 0, (C) score 1+, and (D) score 2+. CCC, clear cell carcinoma; IHC, immunohistochemical; LC3A, light chain 3A; NC, negative control.

• 

  Fig. 2. PFS and OS of the patients with ovarian CCCs according to LC3A expressions. (A) PFS. (B) OS. CCC, clear cell carcinoma; LC3A, light chain 3A; OS, overall survival; PFS, progression-free survival.

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  Fig. 3. Sensitization to cisplatin by autophagy inhibition using HCQ sulfate in ovarian clear cell cancer cell lines. (A) The activity of HCQ as a single agent was measured. (B) Western blotting analysis after treatment by 0 and 25 μ M of HCQ for 24 hours revealed up-regulation of LC3AII, indicating inhibition of autophagy. (C) Cell viability by cisplatin treatment after pre-treatment using 25 μ M of HCQ was measured. (D) Western blotting analysis revealed up-regulation of LC3AII and cleaved-PARP after treatment of 10 nM of cisplatin, with or without 25 μ M of HCQ. Equivalent amounts (10 μ g) of proteins were subjected to SDS-PAGE and blotted with anti-LC3A, anti-PARP, anti-cleaved-PARP, or anti-β-actin antibodies. Cell viability was assessed at 5 days after treatment by MTT assay. HCQ, hydroxychloroquine sulfate; LC3A, light chain 3A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, polymerase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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