Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul 26;7(30):47620-47636.
doi: 10.18632/oncotarget.9951.

Suppression of ABHD2, identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

Koji Yamanoi  1 , Noriomi Matsumura  1 , Susan K Murphy  2 , Tsukasa Baba  1 , Kaoru Abiko  1 , Junzo Hamanishi  1 , Ken Yamaguchi  1 , Masafumi Koshiyama  1 , Ikuo Konishi  1 , Masaki Mandai  3
Affiliations

Suppression of ABHD2, identified through a functional genomics screen, causes anoikis resistance, chemoresistance and poor prognosis in ovarian cancer

Koji Yamanoi et al. Oncotarget. .

Abstract

Anoikis resistance is a hallmark of cancer, and relates to malignant phenotypes, including chemoresistance, cancer stem like phenotypes and dissemination. The aim of this study was to identify key factors contributing to anoikis resistance in ovarian cancer using a functional genomics screen. A library of 81 000 shRNAs targeting 15 000 genes was transduced into OVCA420 cells, followed by incubation in soft agar and colony selection. We found shRNAs directed to ABHD2, ELAC2 and CYB5R3 caused reproducible anoikis resistance. These three genes are deleted in many serous ovarian cancers according to The Cancer Genome Atlas data. Suppression of ABHD2 in OVCA420 cells increased phosphorylated p38 and ERK, platinum resistance, and side population cells (p<0.01, respectively). Conversely, overexpression of ABHD2 decreased resistance to anoikis (p<0.05) and the amount of phosphorylated p38 and ERK in OVCA420 and SKOV3 cells. In clinical serous ovarian cancer specimens, low expression of ABHD2 was associated with platinum resistance and poor prognosis (p<0.05, respectively). In conclusion, we found three novel genes relevant to anoikis resistance in ovarian cancer using a functional genomics screen. Suppression of ABHD2 may promote a malignant phenotype and poor prognosis for women with serous ovarian cancer.

Keywords: anoikis resistance; functional genomics screen; ovarian cancer; shRNA library.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Schematic of functional genomics screens
a. Seven human ovarian serous adenocarcinoma cell lines and an immortalized human ovarian surface cell line HOSE/E7, all of which do not grow in soft agar, were used. Following transfection of the shRNA library, only OVCA420 cells formed colonies in soft agar. 43 colonies were successfully expanded. shRNAs were amplified by PCR and we reconstructed 69 different shRNA plasmids. Out of the 69 shRNAs in OVCA420 cells, 11 again generated colonies in soft agar. We then measured mRNA expression of these 11 genes using RT-PCR. Of the 11 shRNAs, shRNAs directed against ABHD2, CYB5R3 and ELAC2 suppressed target gene mRNA expression. b. Left: shRNA-ABHD2 transfected OVCA420 cell colony in soft agar. Black bar, 100 μm. Right: normalized ABHD2 / ACTB mRNA expression analyzed by RT-PCR. (n=3, respectively) c. Left: shRNA-ELAC2 transfected OVCA420 cell colony in soft agar. Right: normalized ELAC2 / ACTB mRNA expression. d. Left: shRNA-CYB5R3 transfected OVCA420 cell colony in soft agar. Right: normalized CYB5R3 / ACTB expression.
Figure 2
Figure 2. ABHD2 mRNA, protein expression and copy number in clinical specimens
mRNA expression was evaluated using log2 normalized values. a. Comparison of ABHD2 mRNA expression between ovarian cancer tissues and serous borderline tumors (SBT) using gene expression microarray datasets GSE9891 and GSE2109. b. Comparison of ABHD2 mRNA expression between serous adenocarcinoma and non-serous adenocarcinoma in microarray dataset GSE2109. c. Copy number alterations for ABHD2 in TCGA samples. Del; deletion, Amp; Amplification. d. Correlation between ABHD2 copy number and mRNA expression in TCGA specimens. e. Representative ABHD2 immunohistochemistry staining for HGSOC (intensity 0, 1 and 2), normal fallopian tube and SBT are shown. Comparison of H-scores among HGSOC, fallopian tube and SBT. The H-score is calculated as 2x the percentage of the most strongly stained area plus 1x the percentage of the most weakly stained area, imparting a total score ranging from 0 to 200.
Figure 3
Figure 3. ELAC2 mRNA expression and copy number in clinical specimens
mRNA expression was evaluated using the log2 normalized values. a. Comparison of ELAC2 mRNA expression between ovarian cancer tissue and SBT or normal ovarian epithelium from the gene expression microarray datasets GSE9891 and GSE6008. b. Comparison of ELAC2 mRNA expression between serous adenocarcinoma and non-serous adenocarcinoma in microarray dataset KOV-75. c. Copy number alterations at ELAC2 in TCGA specimens. d. Correlation between ELAC2 copy number and mRNA expression in the TCGA dataset.
Figure 4
Figure 4. CYB5R3 mRNA expression and copy number in clinical specimens
mRNA expression was evaluated using the log2 normalized value. a. Comparison of CYB5R3 mRNA expression between ovarian cancer tissue and SBT in the gene expression microarray dataset GSE9891. b. Comparison of CYB5R3 mRNA expression between serous adenocarcinoma and non-serous adenocarcinoma in microarray datasets KOV-75 and GSE2109. c. Copy number alterations of CYB5R3 in TCGA specimens. d. Correlation between CYB5R3 copy number and mRNA expression in the TCGA dataset.
Figure 5
Figure 5. Functional validation of ABHD2 as a negative regulator of anoikis resistance in OVCA420 cells
a. Number of viable control, sh1-OVCA420 and sh2-OVCA420 cells following incubation on ultra-low attachment plates (n=6). b. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates. c. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction (viable cells) and Annexin V(+) fraction (apoptotic cells) between control, sh1 and sh2 cells. d. Number of viable OVCA420-control and OVCA420-ABHD2 cells following incubation on ultra-low attachment plates (n=6). Panels a-c: sh1; sh1-OVCA420, sh2; sh2-OVCA420, control; control-OVCA420; panel d: control; OVCA420-control, ABHD2; SKOV3-ABHD2.
Figure 6
Figure 6. Functional validation of ABHD2 as a negative regulator of anoikis resistance in SKOV3 cells
a. Number of viable SKOV3-control and SKOV3-ABHD2 cells following incubation on ultra-low attachment plates (n=6). b. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates. c. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction and Annexin V(+) fraction between SKOV3-control and SKOV3-ABHD2 cells. d. Number of viable control- SKOV3, sh1- SKOV3-ABHD2 and sh2-SKOV3-ABHD2 cells following incubation on ultra-low attachment plates (n=6). e. Representative data showing Annexin V/7-ADD staining following incubation on ultralow attachment plates for 48 hours. f. Comparison of the ratio of the Annexin V(−)/7-ADD(−) fraction and Annexin V(+) fraction among control, sh1-SKOV3-ABHD2 and sh2-SKOV3-ABHD2 cells. Panels a-c: control; SKOV3-control, ABHD2; SKOV3-ABHD2; panels d-f: control; control-SKOV3-ABHD2, sh1; sh1-SKOV3-ABHD2, sh2; sh2-SKOV3-ABHD2.
Figure 7
Figure 7. Regulation of ERK1/2 and p38MAPK pathways by ABHD2
All experiments were performed in triplicate. a. Phosphorylated p38 (P-P38) and phosphorylated ERK1/2 (P-ERK1/2) increased following knockdown of ABHD2 (sh1 and sh2) in OVCA420 cells. On the contrary, P-P38 and P-ERK1/2 decreased following overexpression of ABHD2 in SKOV3 and OVCA420 cells. b. Resistance of OVCA420 cells to anoikis on ultra-low attachment dishes was inhibited by GSK1120212, a specific inhibitor of the ERK1/2 pathway. Reduction of P-ERK1/2 following treatment with GSK1120212 was confirmed by Western blotting. DMSO, vehicle control. Cells were treated with differing doses of GSK1120212 as indicated. c. Resistance of OVCA420 cells to anoikis was inhibited following treatment with SB203580, a specific inhibitor of the the p38MAPK pathway. d. Levels of P-P38 and P-ERK1/2 increased following knockdown of ABHD2 (sh1 and sh2) in SKOV3-ABHD2 cells. e. Resistance to anoikis in sh1-SKOV3-ABHD2 and sh2-SKOV3-ABHD2 cells was inhibited following treatment with 100nM GSK1120212 (GSK) and 30μM SB203580 (SB).”
Figure 8
Figure 8. Overall survival of ovarian cancer patients
a. Differences in survival based on ABHD2 immunohistochemical scores (H-score) in HGSOC. b. Differences in survival based on ABHD2 mRNA expression in GSE9891 (n=285, mostly HGSOC) and GSE3149 (n=146, mostly HGSOC) datasets. Samples were divided into high (greater than the median value) and low (less than the median) expression cases. c. Analysis of HGSOC patients (n=36) from KOV-75 based on ABHD2 mRNA expression. d. Analysis of non-HGSOC patients (n=39) from KOV-75 based on ABHD2 mRNA expression. n.s., not significant.
Figure 9
Figure 9. Suppression of ABHD2 causes platinum resistance
a. Representative data showing 7-ADD staining following 24 hour incubation with 10 μM cisplatin. b. The ratio of the 7-AAD negative live OVCA420 cells markedly increased following suppression of ABHD2 (sh1 and sh2) as compared to the control after 24 hour incubation with 10 μM cisplatin (n=3). c. Dose-response curves following incubation of OVCA420 cells with the indicated concentrations of cisplatin for 72 hours (n=6). d. Cisplatin IC50 values increased following suppression of ABHD2 in OVCA420 cells. e. Representative data showing 7-ADD staining following 24 hour incubation with 100 μM Carboplatin. f. The ratio of 7-AAD negative live OVCA420 cells markedly increased following suppression of ABHD2 (sh1 and sh2) as compared to the control following a 24 hour incubation with 100 μM carboplatin (n=3).
See this image and copyright information in PMC

References

    1. Coleman RL, Monk BJ, Sood AK, Herzog TJ. Latest research and treatment of advanced-stage epithelial ovarian cancer. Nat Rev Clin Oncol. 2013;10:211–24. - PMC - PubMed
    1. Seidman JD, Horkayne-Szakaly I, Haiba M, Boice CR, Kurman RJ, Ronnett BM. The histologic type and stage distribution of ovarian carcinomas of surface epithelial origin. Int J Gynecol Pathol. 2004;23:41–4. - PubMed
    1. Cancer Genome Atlas Research Network Integrated genomic analyses of ovarian carcinoma. Nature. 2011;474:609–15. - PMC - PubMed
    1. Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol. 1994;124:619–26. - PMC - PubMed
    1. Carduner L, Picot CR, Leroy-Dudal J, Blay L, Kellouche S, Carreiras F. Cell cycle arrest or survival signaling through alphav integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids. Exp Cell Res. 2014;320:329–42. - PubMed