Select Page

RESEARCH ARTICLE

PDF logo-blue 30

Elimination of Sestrin 2 Compromises Viability in Extracellular Matrix-Detached SKOV3 Ovarian Cancer Cells

Yadira Gonzalez, Leann Tulisiak, Mary Philbin, Hannah Voss, Ethan Schramm, Irma Ruelas, and Calli A. Davison-Versagli 

Department of Biology, Saint Mary’s College, Notre Dame, IN 46556, USA 

Correspondence: cversagli@saintmarys.edu (C.A.D-V.) 

Gonzalez Y et al. Reactive Oxygen Species 8(24):332–340, 2019; ©2019 Cell Med Press

http://dx.doi.org/10.20455/ros.2019.861

(Received: July 10, 2019; Revised: July 22, 2019; Accepted: July 23, 2019) 

ABSTRACT | Epithelial ovarian carcinoma (EOC) is considered the deadliest gynecological cancer, largely due to the fact that it is often diagnosed once the cancer has already metastasized, thus making the disease more difficult to treat. Throughout metastasis, ovarian epithelial cancer cells must overcome many feats, including surviving in extracellular matrix (ECM) detachment. ECM-detached cancer cells must evade a number of insults, including increased intracellular reactive oxygen species (ROS). Recent evidence suggests ECM-detached cancer cells rely on antioxidant enzymes to combat these increasing levels of ROS to promote survival; however, the specific antioxidant enzymes involved in this process have yet to be fully elucidated. Sestrin 2 (SESN2) is a multi-functional protein that has been found to be instrumental in many different signaling pathways; notably, it has been recognized to play a critical role in eliminating ROS. Here, we show that SESN2 plays a unique role in maintaining the viability of ECM-detached metastatic ovarian epithelial cancer cells, and elimination of this critical protein results in compromised viability. Thus, these data identify SESN2 as a potentially interesting therapeutic target for treating this deadly metastatic disease. 

KEYWORDS | Extracellular matrix; Ovarian cancer; Reactive oxygen species; Sestrin 

ABBREVIATIONS | ECM, extracellular matrix; EOC, epithelial ovarian carcinoma; ROS, reactive oxygen species; SESN2, sestrin 2; shRNA, short hairpin RNA 

CONTENTS 

  1. Introduction
  2. Materials and Methods

2.1. Cell Culture

2.2. Short Hairpin RNA (shRNA)

2.3. Western Blotting

2.4. Soft Agar Assay

2.5. Cell Proliferation and Cell Viability Assay

2.6. Statistical Analysis

  1. Results

3.1. SESN2 Deficiency Decreases Survival of Anchorage-Independent SKOV3 Cells

3.2. SESN2 Deficiency Compromises Cell Viability in ECM-Detached SKOV3 Cells

3.3. Attached SESN2-Deficient SKOV3 Cells Sustained Cell Viability and Proliferation

  1. Discussion
  2. Conclusion

1. INTRODUCTION 

Epithelial ovarian carcinoma (EOC) is the most common form of ovarian cancer and is often more aggressive than nonepithelial malignancies [1]. Given their aggressive nature, 65% of EOC cases are diagnosed after the disease has already metastasized (or spread) to other areas throughout the abdominal cavity or to distant organs, thus making the disease more difficult to treat [1]. It has become clear that better therapeutic options are needed to remedy patients that present with this disease. During metastasis, EOC cells escape the ovaries and use the peritoneal fluid to travel to organs throughout the abdominal cavity before eventually utilizing the bloodstream and lymphatics to travel to distant areas, including the skin, lungs, and brain [2]. Throughout this process, EOC cells must gain the ability to thrive while being detached from the extracellular matrix (ECM) [3‒5]. It has been recognized that ECM-detached breast cancer cells must overcome many ECM detachment-induced insults, including increases in reactive oxygen species (ROS) [3, 4]. Specifically, ECM-detached breast cancer cells combat increases in ROS through utilizing antioxidant enzymes, a family of enzymes that facilitate oxidant scavenging [3]. However, the importance of antioxidant enzymes in the successful metastasis of EOC cells has yet to be fully elucidated. 

Sestrin 2 (SESN2) is a member of a family of proteins (SESN1-3) that are highly conserved and expressed in humans [6]. While this stress-inducible protein appears to lack intrinsic catalytic antioxidant activity, SESN2 is involved in processes that decrease ROS levels [7]. Specifically, SESN2 promotes oxidoreductase activity and mediates the expression of Nrf2, a transcription factor responsible for controlling antioxidant enzyme expression [7, 8]. Most recently, SESN2 has been found to be implicated in cancer. Decreased SESN2 expression in colorectal cancer tissues has been connected with poor prognosis [9]. Furthermore, increased SESN2 expression has been connected with successful ionizing radiation-inducing killing of breast cancer cells [10]. While recent studies have begun to unravel the role of SESN2 in tumorigenesis, SESN2 and its importance in EOC, specifically its importance in anchorage independence and metastasis, has yet to be explored. 

Here, we sought to determine the role of SESN2 in the viability and proliferation of ECM-detached SKOV3 cells, an EOC cell line. We discovered that SESN2 uniquely protects the viability of anchorage-independent SKOV3 cells, and elimination of SESN2 in ECM-detached SKOV3 cells results in substantial cell death. In aggregate, these data define SESN2 as a new potential therapeutic target for metastatic EOC.

2. MATERIALS AND METHODS 

2.1. Cell Culture 

SKOV3 ovarian cancer cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in McCoy’s 5A media modified with L-glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS) (Alkali Scientific, Ocala, FL, USA), and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). The cells were maintained at 37°C and 5% CO2. 

2.2. Short Hairpin RNA (shRNA) 

MISSION shRNA targeting SESN2 was purchased from Sigma-Aldrich, and stable knockdown of SESN2 was achieved using lentiviral transduction as described previously [11]. 

2.3. Western Blotting 

Western blotting was performed as described previously [11]. To confirm SESN2 knockdown, SKOV3 parental and SKOV3 SESN2 cells were lysed in 1% NP-40 (Sigma-Aldrich) with aprotinin (1 mg/ml) (Gibco) and leupeptin (5 mg/ml) (Gibco) for 20 min, spun at 18,188 g for 30 min at 4oC, and normalized using a BCA assay (Pierce Biotech, Rockford, IL, USA). Laemmeli loading dye (Bio-Rad, Hercules, CA, USA) was added to normalized lysates and lysates were run at 150 V on a 12% SDS-PAGE gel (Bio-Rad) before being transferred to PVDF membrane (Millipore, St. Louis, MO, USA) at 100 V. The membrane was blocked in 5% milk in 1× TBST (Bio-Rad) for 30 min and incubated with the primary antibodies used at a dilution of 1:1000 for rabbit anti-SESN2 (cat # 10795-1-AP, Proteintech, Rosemont, IL, USA) and mouse anti-b actin (cat # 66009-1-Ig, Proteintech) as a loading control overnight. The blot was washed with 1× TBST (Bio-Rad) three times for 20 min before the goat anti-rabbit HRP-conjugated (cat # SA00001-2, Proteintech) and goat anti-mouse HRP-conjugated (cat# SA00001-1, Proteintech) secondary antibodies were used at a dilution of 1:1500 in 5% milk and 1× TBST. The membrane was washed three times for 10 minutes using 1× TBST before being developed using chemiluminescence (Alkali Scientific).

2.4. Soft Agar Assay

The soft agar assay was completed as described previously [11]. Briefly, a mixture of 0.4% agarose and media (2 ml) were poured into each well of a six-well plate. SKOV3 parental cells or SKOV3 SESN2-deficient cells were plated in triplicate on the six-well plate at 20,000 cells per well in 1.5 ml of 0.5% agarose (Sigma-Aldrich) and were plated on top of cooled 0.4% media and agarose wells. Plates were incubated for 21 days at 37oC and 5% CO2 and wells were fed with 1 ml of media every two days. This experiment was repeated at least three times, and colonies were counted per well using ImageJ software (NIH, Bethesda, MD, USA). 

2.5. Cell Proliferation and Cell Viability Assay 

Cell proliferation and viability in ECM attachment were determined by using trypan blue exclusion as described before [11]. Approximately 50,000 cells were plated in 1 ml of media per well in triplicate for SKOV3 parental and SKOV3 SESN2-deficient cells in ECM attachment using six-well tissue culture plates (Cytone 1, Ocala, Florida, USA) or in ECM detachment using poly-HEMA-coated plates (Cytone 1). After 24, 48, and 72 h, cells were collected, stained with trypan blue (Hyclone, Logan, UT, USA), and total cells and total dead cells were counted using a hemocytometer. This experiment was repeated three times for ECM-attached cells and ECM-detached cells. 

2.6. Statistical Analysis 

Statistical significance was determined using a two-tailed t-test for soft agar, cell proliferation, and cell viability assays where p < 0.05 was considered statistically significant. Error bars represent standard error of the mean (SEM).

3. RESULTS 

3.1. SESN2 Deficiency Decreases Survival of Anchorage-Independent SKOV3 Cells 

The soft agar assay tests the ability of cancer cells to form colonies in anchorage-independent environments similar to what is encountered during metastasis. To determine if SESN2 deficiency affected anchorage-independent growth of SKOV3 cells, SKOV3 cells were engineered to be deficient in SESN2 using shRNA techniques, and SESN2 deficiency was confirmed by western blot (Figure 1A). The soft agar assay was then used to determine if SESN2 deficiency affected anchorage-independent colony growth when compared to SKOV3 parental cells. Results showed that SESN2-deficient SKOV3 cells had significantly less colony formation than parental cells (Figure 1Band 1C).

 

FIGURE 1. SESN2 deficiency in SKOV3 cells severely abrogates growth in soft agar. SESN2 deficiency was achieved in SKOV3 cells utilizing shRNA techniques and successful elimination was confirmed through western blotting (A). SKOV3 parental and SKOV3 SESN2-deficient cells were plated in triplicate in soft agar, fed every two days with 1 ml of media, and grown for 21 days. After 21 days, colonies were stained with iodonitrotetrazolium chloride (INT) and colonies were quantified using ImageJ software. Representative images from one individual experiment are in (B) and quantitation of colony number is shown in (C). Error bars represent SEM. *, p< 0.05. 

3.2. SESN2 Deficiency Compromises Cell Viability in ECM-Detached SKOV3 Cells 

In order to determine if lack of colony formation in soft agar in SESN2-deficient cells was due to decreases in proliferation or changes in cell viability, SKOV3 parental cells and SKOV3 SESN2-deficient cells were plated on poly-HEMA-coated plates. Poly-HEMA plates do not allow for cells to deposit matrix; therefore, cells are grown in ECM detachment. When plated in detachment, SKOV3 SESN2-deficient cells displayed decreased cell viability at the 48- and 72-hperiods when comparedto the SKOV3 parental cells (Figure 2A). However, there was no change in proliferation between ECM-detached SKOV3 parental cells and ECM-detached SKOV3 SESN2-deficient cells (Figure 2B). These data suggest that abrogated anchorage-independent growth visualized in SESN2-deficient SKOV3 cells is due to compromised cell viability rather than cell proliferation.

 

FIGURE 2. Elimination of SESN2 compromises cell viability in ECM-detached SKOV3 cells. SKOV3 parental and SKOV3 SESN2-deficient cells were plated in triplicate on non-adherent poly-HEMA-coated plates, and cells were analyzed at the 24, 48, and 72-h time points. This experiment was completed three times. Percent cell viability calculated at 24, 48, and 72-h time points is represented graphically in (A), where vertical bars represent the mean of all three experiments. Total proliferation at all three time points is shown in (B), and vertical bars represent the mean of all three experiments. Error bars represent the SEM. *, p< 0.05. 

3.3. Attached SESN2-Deficient SKOV3 Cells Sustained Cell Viability and Proliferation 

Noting that SESN2 deficiency abrogated cell viability in ECM-detached SKOV3 cells, we were interested to see if SESN2 deficiency affected cell viability and/or cell proliferation in ECM-attached SKOV3 cells. SKOV3 parental cells and SKOV3 SESN2-deficient cells were plated on adherent 6-well plates, and counted at 24, 48, and 72-h time points. Interestingly, SESN2 deficiency had no effect on cell viability (Figure 3A) or cell proliferation (Figure 3B) in ECM-attached SKOV3 cells. All together, these results indicate that SESN2 deficiency has little impact on ECM-attached SKOV3 cells, suggesting that SESN2 deficiency plays little role in proliferation and cell viability at the primary tumor and a larger role during metastasis.

 

FIGURE 3. Elimination of SESN2 does not impact proliferation or cell viability of ECM-attached SKOV3 cells. SKOV3 parental and SKOV3 SESN2-deficient cells were plated on adherent plates in triplicate and counted after 24, 48, and 72 h. Percent viability is shown in (A) and total proliferation is represented graphically in (B). In both graphs, vertical bars represent the mean of three separate experiments and the error bars indicate SEM. No statistical significance was seen between ECM-attached SKOV3 parental cells and ECM-attached SKOV3 SESN2-deficient cells at any time point for cell viability or cell proliferation.

4. DISCUSSION 

Here, our studies work to unravel the importance of SESN2 in the viability and proliferation in anchorage-independent SKOV3 epithelial ovarian cancer cells. We report that SESN2 plays a critical role in the colony formation of SKOV3 cells in soft agar. Specifically, SESN2 plays a unique and important role in maintaining cell viability in ECM-detached SKOV3 cells. Interestingly, SESN2 deficiency in ECM-attached cells displays no significant change in cell viability. Furthermore, SESN2 appears to play no role in maintaining proliferation in ECM-detached or ECM-attached SKOV3 cells (Figure 4).

 

FIGURE 4. A proposed model for the role of SESN2 in SKOV3 epithelial ovarian cancer cells. Here, we report that while SESN2 deficiency has no impact on ECM-attached epithelial ovarian cancer cells at the primary tumor, SESN2 deficiency in ECM-detached epithelial ovarian cancer cells results in severely abrogated cell viability. Given that survival in ECM detachment is necessary for metastasis, these data indicate that SESN2 expression is important for maintaining cell viability and is important for successful metastasis of epithelial ovarian cancer cells. Therefore, SESN2 expression is critical for survival and metastasis of epithelial ovarian cancer cells. 

The identification of SESN2 as key protein important in the viability of ECM-detached ovarian cancer cells adds to a growing body of literature that ROS-eliminating mechanisms are critical in metastatic cancers. Most recently, the antioxidant enzyme, catalase, was found to play a critical role in the viability of ECM-detached breast cancer cells and ECM-detached ovarian cancer cells [3, 11]. Furthermore, superoxide dismutase 2 (SOD2) has been implicated in breast cancer and nasopharyngeal anchorage-independent growth and metastasis [3, 12]. Here, we report that SESN2 plays a critical role in the viability of anchorage-independent EOC cells. Thus, novel therapeutic strategies aimed at SESN2 may be an effective strategy to eliminate ECM-detached metastatic EOC cells. 

Our study is the first (to our knowledge) to study SESN2 in EOC and directly link SESN2 and the survival of ECM-detached EOC cells. The vast majority of studies have been aimed at understanding the role of SESN2 in basic physiological processes [13‒18]; however, only recently has SESN2 been studied in cancer. As of late, it has been reported that decreased SESN2 expression has been connected to poor outcome in colorectal cancer [9], and induction of SESN2 through a variety of mechanisms has been connected with successful elimination of cancer cells [10, 19‒22]. Interestingly, our study finds that elimination of SESN2 compromises viability of ECM-detached metastatic EOC cells. Thus, our study represents a novel paradigm whereby SESN2 expression actually promotes survival of ECM-detached metastatic EOC cells. In hepatocellular carcinoma, SESN2 expression was increased compared to the benign tissue, and this increased SESN2 expression was connected with resistance to the first-line therapy sorafenib [23]. Collectively, these studies reveal that the role of SESN2 is unique in many different contexts, and future studies aimed at elucidating the role of SESN2 in different contexts are warranted to better treat this disease. 

In aggregate, it is plausible that SESN2 elimination could be a viable therapeutic tactic for specifically treating metastatic EOC. Given the low 5-year survival rate associated with late-stage cancer, it is crucial to work to develop better treatments for patients diagnosed with this disease [1]. Furthermore, it is of critical importance to study SESN2 expression levels in EOC tissues to determine if SESN2 could serve as an improved diagnostic biomarker.

5. CONCLUSION 

Here, we report SESN2 deficiency compromises growth of ECM-detached SKOV3 EOC cells in the soft agar assay. Furthermore, this decreased colony formation is the result of decreased viability in SESN2-deficient SKOV3 cells. Interestingly, elimination of SESN2 in ECM-attachedcells showed no changes in proliferation or viability, suggesting that SESN2 plays a unique and critical role in the growth of ECM-detached SKOV3 cells. Therefore, SESN2 is a plausible target for treating metastatic EOC and requires further investigation. 

ACKNOWLEDGMENTS 

We would like to thank Dr. Zachary Schafer and his entire lab for allowing us to use his laboratory space while our laboratory was under construction. We would also like to thank the Helen Kuhn Carey research fund and the Biology Department of Saint Mary’s College for funding this work. The authors declare no conflicts of interest. 

REFERENCES 

  1. Torre LA, Trabert B, DeSantis CE, Miller KD, Samimi G, Runowicz CD, et al. Ovarian cancer statistics, 2018. CA Cancer J Clin 2018; 68(4):284‒96. doi: 10.3322/caac.21456.
  2. Langyel E. Ovarian cancer development and metastasis. Am J Pathol 2010; 177(3):1053‒64. doi: 10.2353/ajpath.2010.100105.
  3. Davison CA, Durbin SM, Thau MR, Zellmer VR, Chapman SE, Diener J, et al. Antioxidant enzymes mediate survival of breast cancer cells deprived of extracellular matrix. Cancer Res 2013; 73(12):3704‒15. doi: 10.1158/0008-5472.can-12-2482.
  4. Schafer ZT, Grassian AR, Song LL, Jiang ZY, Gerhart-Hines Z, Irie HY, et al. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 2009; 461(7260):109‒U18. doi: 10.1038/nature08268.
  5. Cai Q, Yan L, Xu Y. Anoikis resistance is a critical feature of highly aggressive ovarian cancer cells. Oncogene 2015; 34(25):3315-24. doi: 10.1038/onc.2014.264.
  6. Lee JH, Budanov AV, Talukdar S, Park EJ, Park HL, Park HW, et al. Maintenance of metabolic homeostasis by sestrin2 and sestrin3. Cell Metab 2012; 16(3):311‒21. doi: 10.1016/j.cmet.2012.08.004.
  7. Rhee SG, Bae SH. The antioxidant function of sestrins is mediated by promotion of autophagic degradation of Keap1 and Nrf2 activation and by inhibition of mTORC1. Free Radic Biol Med 2015; 88(Pt B):205‒11. doi: 10.1016/j.freeradbiomed.2015.06.007.
  8. Bae SH, Sung SH, Oh SY, Lim JM, Lee SK, Park YN, et al. Sestrins activate Nrf2 by promoting p62-dependent autophagic degradation of Keap1 and prevent oxidative liver damage. Cell Metab 2013; 17(1):73‒84. doi: 10.1016/j.cmet.2012.12.002.
  9. Wei JL, Fu ZX, Fang M, Guo JB, Zhao QN, Lu WD, et al. Decreased expression of sestrin 2 predicts unfavorable outcome in colorectal cancer. Oncol Rep 2015; 33(3):1349‒57. doi: 10.3892/or.2014.3701.
  10. Sanli T, Linher-Melville K, Tsakiridis T, Singh G. Sestrin2 modulates AMPK subunit expression and its response to ionizing radiation in breast cancer cells. PLoS One 2012; 7(2):e32035. doi: 10.1371/journal.pone.0032035.
  11. Libbing CL, Jungles KM, Rabaut E, Davison-Versagli CA. Catalase deficiency compromises survival in extracellular matrix-detached SKOV3 ovarian cancer cells. React Oxyg Species (Apex) 2019; 7(20):121‒8. doi: 10.20455/ros.2019.821.
  12. Li S, Mao Y, Zhou T, Luo C, Xie J, Qi W, et al. Manganese superoxide dismutase mediates anoikis resistance and tumor metastasis in nasopharyngeal carcinoma. Oncotarget 2016; 7(22):32408‒20. doi: 10.18632/oncotarget.8717.
  13. Ishihara M, Urushido M, Hamada K, Matsumoto T, Shimamura Y, Ogata K, et al. Sestrin-2 and BNIP3 regulate autophagy and mitophagy in renal tubular cells in acute kidney injury. Am J Physiol Renal Physiol 2013; 305(4):F495‒509. doi: 10.1152/ajprenal.00642.2012.
  14. Kallenborn-Gerhardt W, Lu R, Syhr KM, Heidler J, von Melchner H, Geisslinger G, et al. Antioxidant activity of sestrin 2 controls neuropathic pain after peripheral nerve injury. Antioxid Redox Signal 2013; 19(17):2013‒23. doi: 10.1089/ars.2012.4958.
  15. Soontornniyomkij V, Soontornniyomkij B, Moore DJ, Gouaux B, Masliah E, Tung S, et al. Antioxidant sestrin-2 redistribution to neuronal soma in human immunodeficiency virus-associated neurocognitive disorders. J Neuroimmune Pharmacol 2012; 7(3):579‒90. doi: 10.1007/s11481-012-9357-0.
  16. Tayyar AT, Tayyar A, Kozali S, Karakus R, Eser A, Abide Yayla C, et al. Maternal serum sestrin 2 levels in preeclampsia and their relationship with the severity of the disease. Hypertens Pregnancy 2019; 38(1):13‒9. doi: 10.1080/10641955.2018.1540702.
  17. Thamsen M, Kumsta C, Li F, Jakob U. Is overoxidation of peroxiredoxin physiologically significant? Antioxid Redox Signal 2011; 14(4):725‒30. doi: 10.1089/ars.2010.3717.
  18. Wang M, Xu Y, Liu J, Ye J, Yuan W, Jiang H, et al. Recent insights into the biological functions of sestrins in health and disease. Cell Physiol Biochem 2017; 43(5):1731‒41. doi: 10.1159/000484060.
  19. Hua X, Xu J, Deng X, Li J, Zhu DQ, Zhu J, et al. New compound ChlA-F induces autophagy-dependent anti-cancer effect via upregulating Sestrin-2 in human bladder cancer. Cancer Lett 2018; 436:38‒51. doi: 10.1016/j.canlet.2018.08.013.
  20. Li DD, Sun T, Wu XQ, Chen SP, Deng R, Jiang S, et al. The inhibition of autophagy sensitises colon cancer cells with wild-type p53 but not mutant p53 to topotecan treatment. PLoS One 2012; 7(9):e45058. doi: 10.1371/journal.pone.0045058.
  21. Liang Y, Zhu J, Huang H, Xiang D, Li Y, Zhang D, et al. SESN2/sestrin 2 induction-mediated autophagy and inhibitory effect of isorhapontigenin (ISO) on human bladder cancers. Autophagy 2016; 12(8):1229‒39. doi: 10.1080/15548627.2016.1179403.
  22. Won DH, Chung SH, Shin JA, Hong KO, Yang IH, Yun JW, et al. Induction of sestrin 2 is associated with fisetin-mediated apoptosis in human head and neck cancer cell lines. J Clin Biochem Nutr 2019; 64(2):97‒105. doi: 10.3164/jcbn.18-63.
  23. Dai J, Huang Q, Niu K, Wang B, Li Y, Dai C, et al. Sestrin 2 confers primary resistance to sorafenib by simultaneously activating AKT and AMPK in hepatocellular carcinoma. Cancer Med 2018; 7(11):5691‒703. doi: 10.1002/cam4.1826.