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

Morikazu Miyamoto; Masashi Takano; Tadashi Aoyama; Hiroaki Soyama; Tomoyuki Yoshikawa; Hitoshi Tsuda; Kenichi Furuya

doi:10.3802/jgo.2017.28.e33

Journal List > J Gynecol Oncol > v.28(3) > 1093818

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Miyamoto, Takano, Aoyama, Soyama, Yoshikawa, Tsuda, and Furuya: Inhibition of autophagy protein LC3A as a therapeutic target in ovarian clear cell carcinomas

Original Article

J Gynecol Oncol 2017;28(3):e33.

Published online: 4 May 2017

DOI: https://doi.org/10.3802/jgo.2017.28.e33

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

Morikazu Miyamoto¹, Masashi Takano^2,, Tadashi Aoyama¹, Hiroaki Soyama¹, Tomoyuki Yoshikawa², Hitoshi Tsuda³, Kenichi Furuya¹

¹Department of Obstetrics and Gynecology, National Defense Medical College Hospital, Tokorozawa, Japan

²Department of Clinical Oncology, National Defense Medical College Hospital, Tokorozawa, Japan

³Department of Basic Pathology, National Defense Medical College Hospital, Tokorozawa, Japan

Correspondence to Masashi Takano Department of Clinical Oncology, National Defense Medical College Hospital, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: mastkn@ndmc.ac.jp

Received 27 November 2016 Revised 17 January 2017 Accepted 31 January 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Ovarian Neoplasms, Adenocarcinoma, Clear Cell, Autophagy, Prognosis

References

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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.

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.

Table 1.

Characteristics of the patients with ovarian CCCs according to LC3A expression levels

Variables Age (yr)	LC3A high expression (n=78)52.5 (32–72)	LC3A low expression (n=22) 50.5 (36–71)	p-value 0.620
FIGO stage			0.060
I	36 (46)	16 (73)
II	8 (10)	3 (14)
III	31 (40)	2 (9)
IV	3 (4)	1 (4)
Lymph node metastasis			0.500
Positive	13 (17)	2 (9)
Negative	41 (53)	15 (68)
Not evaluated	24 (30)	5 (23)
Residual tumor at primary surgery			0.220
Optimal	62 (80)	20 (91)
Suboptimal	16 (20)	2 (9)
Response to primary chemotherapy in patients with evaluable disease			<0.010
CR	3 (13)	3 (75)
PR	1 (4)	1 (25)
SD	1 (4)	0 (0)
PD	19 (79)	0 (0)
Response rate (%)	17	100

Values are presented as median (range) or number (%). CCC, clear cell carcinoma; CR, complete response; FIGO, the International Federation of Gynecology and Obstetrics; LC3A, light chain 3A; PD, progressive disease; PR, partial response; SD, stable disease.

Table 2.

Multivariate analyses for PFS and OS in the patients with ovarian CCCs

Variables		PFS			OS
Variables		HR	95% CI	p-value	HR	95% CI	p-value
Age (yr)	>51 vs. ≤50	1.11	0.61–2.09	0.730	0.85	0.44–1.65	0.620
FIGO stage	III/IV vs. I/II	4.58	2.44–8.77	<0.010	3.84	1.82–8.42	<0.010
Residual tumor at primary surgery	Suboptimal vs. optimal	3.51	1.67–7.11	<0.010	5.02	2.28–10.80	<0.010
LC3A expression	High expression vs. low expression	2.64	1.17–7.12	0.020	4.45	1.53–19.00	<0.010

CCC, clear cell carcinoma; CI, confidence interval; FIGO, the International Federation of Gynecology and Obstetrics; HR, hazard ratio; LC3A, light chain 3A; OS, overall survival; PFS, progression-free survival.

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