The PI3K/mTOR dual inhibitor GSK458 potently impedes ovarian cancer tumorigenesis and metastasis

Yangjiong Xiao1,2,3,4 • Yang Yu4 • Pengcheng Jiang5 • Yuhong Li6 • Chao Wang6 • Rong Zhang 1

Received: 6 December 2019 / Revised: 30 March 2020 / Accepted: 3 April 2020
Ⓒ International Society for Cellular Oncology 2020

Purpose The PI3K/AKT/mTOR pathway is one of the most highly activated cellular signaling pathways in advanced ovarian cancer. Although several PI3K/AKT/mTOR inhibitors have been developed to treat various types of cancer, the antitumor efficacy of many of these compounds against ovarian cancer has remained unclear.
Methods Here, we tested and compared a panel of 16 PI3K/AKT/mTOR inhibitors (XL765, Miltefosine, Rapamycin, CCI-779, RAD001, FK506, XL147, GSK2110183, IPI-145, GSK2141795, BYL719, GSK458, CAL-101, XL765 analogue SAR245409,
Triciribine, and GDC0941) that have entered clinical trials for antitumor activity against ovarian cancer, as well as the front line drug, paclitaxel. Antitumor efficacy was measured in both ovarian cancer cell lines and patient-derived ovarian primary tumor cell lines in vitro and in vivo.
Results We identified the PI3K/mTOR dual inhibitor GSK458 as a potent inhibitor of proliferation in all cell lines tested at half maximal inhibitory concentrations (IC50) of approximately 0.01-1 µM, a range tens to hundreds fold lower than that of the other PI3K/AKT/mTOR inhibitors tested. Additionally, GSK458 showed the highest inhibitory efficacy against ovarian cancer cell migration. GSK458 also inhibited tumor growth and metastasis in nude mice intraperitoneally engrafted with SKOV3 cells or a patient-derived tumor cell xenograft (PDCX). Importantly, the inhibitory efficiency of GSK458 on cell proliferation and migra- tion both in vitro and in vivo was comparable to that of paclitaxel. Mechanistically, the anti-tumor activity of GSK458 was found to be associated with inactivation of AKT and mTOR, and induction of cell cycle arrest at the G0/G1 phase.
Conclusions Based on our results, we conclude that GSK458 may serve as an attractive candidate to treat ovarian cancer.

Keywords Ovarian cancer . PI3K/AKT/mTOR inhibitor . GSK458 . Xenograft . Primary cell lines . Target therapy

⦁ Introduction

Over the past two decades, the five-year overall survival (OS) rate for ovarian cancer patients has remained low (30% − 40%).
There were an estimated 295,414 cases and 184,799 deaths of ovarian cancer around the world in 2018 [1]. Although the ovarian cancer death rate has plateaued over the last two de- cades, it remains high worldwide, making ovarian cancer the

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⦁ Yangjiong Xiao ⦁ [email protected]
⦁ Rong Zhang ⦁ [email protected]

1 Department of Obstetrics and Gynecology, Shanghai Fengxian District Central Hospital, Southern Medical University, 201499 Shanghai, China
2 State Key Laboratory of Respiratory Disease, Guangzhou Medical University, 510182 Guangzhou, China
3 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
4 Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University, Shanghai 200241, China
5 Department of Gynecology, Changzhou Second People’s Hospital Affiliated to Nanjing Medical University, Changzhou 213004, China
6 Department of Gynecology, The International Peace Maternity & Child Health Hospital, The China Welfare Institute, Shanghai Jiaotong University, Shanghai 200030, China

deadliest tumor among all gynecological malignancies [2–4]. As symptoms of ovarian cancer are inapparent at early stages, about two-thirds of patients are at an advanced disease stage at diagnosis. Several treatments have entered the clinic. Carboplatin and paclitaxel exhibit excellent antitumor effects as first-line treatments, however, around two-thirds of the pa- tients become resistant to these drugs and ultimately relapse [5]. Several targeted (poly ADP ribose polymerase (PARP) inhibi- tors) and immuno-therapy (anti-PD-1 and PD-L1 antibodies) strategies have been evaluated, with only modestly increased OS rates (2%-4%) [6]. Although PARP inhibitors (Olaparib, Rucaparib or Niraparib) can increase progression-free survival (PFS) durations of ~ 5 to 16 months in patients with or without BRCA1/2 mutations, they only marginally improve the five- year OS. In phase 2 and phase 3 clinical trials, for example, the reported OS was only prolonged for about 2 to 5 months by Olaparib therapy (58% and 24% data maturity, respectively) [7, 8]. Anti-PD-1 and/or PDL-1 antibodies are two of the most successful immuno-therapeutics presently used in the clinic [9]. However, only 6–15% of ovarian cancer patients respond to PD-1/PDL-1 blockade, with a median PFS of anti-PD-1 or PDL-1 antibody treatment being only ~ 6 months [10, 11]. Therefore, novel effective treatment strategies are urgently needed to improve the OS of ovarian cancer patients [5].
In this study, we found that the phosphatidylinositol 3-kinase (PI3K)/AKT pathway is highly activated in advanced (stage III and IV) compared to early stage (stage II) ovarian tumors by KEGG pathway enrichment analysis of mRNA sequencing da- ta from The Cancer Genome Atlas (TCGA) database. This observation suggests that the PI3K/AKT signaling pathway may play a critical role in ovarian cancer metastasis and recur- rence. Consistent with our observation, previous studies have shown that the PI3K/AKT pathway may be an attractive ther- apeutic target for treating ovarian cancer patients [12, 13]. Also, the TCGA consortium reported that the PI3K/AKT/mTOR (mammalian target of rapamycin, mTOR) pathway was activat- ed in 34% of the tumors from 316 high grade serous ovarian cancer patients [14]. PI3K/AKT/mTOR is one of the most stud- ied signaling pathways in cancer biology because of its wide involvement in carcinogenesis, metastasis, recurrence and che- motherapeutic drug resistance [15]. It has been reported that the PI3K/AKT/mTOR pathway is highly activated in approximate- ly 70% of ovarian cancers [16, 17]. Several PI3K/AKT/mTOR inhibitors have shown some therapeutic effects against ovarian cancers, alone or in combination with other drugs such as pac- litaxel or carboplatin, in clinical trials [15, 16]. Among the most widely studied PI3K/AKT/mTOR inhibitors are the PI3K in- hibitors (BKM120 [18], XL147, GDC0941, PX866, ETP-
46321 [19], and CH5132799 [20]), AKT inhibitors (Perifosine, MK2206, GSK2141795 [16] and GSK690693),
mTOR inhibitors (Rapamycin, RAD001 [21], CCI-779 [16, 22–24], Ridaforolimus, AZC8055, OSI-027, AZD2014 [25]
and PP242 [26]), PI3K/mTOR dual inhibitors (SF1126 [16],
GSK458 [16], XL765 [16], PF-04691502 [27], PF-05212384
[27], BEZ235 [28] and DS-7423 [29]) and the mTOR/AKT
dual inhibitor (MKC1 [16]). However, in most studies these inhibitors only showed modest antitumor activity. In addition, many of them were reported in independent publications mak- ing it difficult to compare them side by side. Most importantly, the effects of these PI3K/AKT/mTOR inhibitors on ovarian cancer have not been elucidated yet, which may be due to study limitations such as limited numbers of cases included or high toxicities of the inhibitors in clinical trials.
Patient-derived tumor xenograft (PDX) and patient-derived tumor cell xenograft (PDCX) mouse models have been shown to replicate the tumor biology of patients [30–32]. Accordingly, our previous work showed similar morphologies (i.e., glands, papillae, stromal cores, and desmoplastic stroma) between xenograft models and patient tumor tissues [33]. Here, we compared, in parallel, the antitumor effects of 16 PI3K/AKT/mTOR inhibitors and paclitaxel, not only in ovar- ian cancer cell lines but also in primary tumor cells in vitro. Also, we tested the effects of high potent candidates in PDX and PDCX models in vivo. Surprisingly, we found that many of the tested inhibitors had little or even no inhibitory effect on ovarian cancer cell proliferation or migration. GSK458, also known as GSK2126458, exhibited the highest anti- proliferative and -migrative activities in both ovarian cancer cell lines and patient-derived primary tumor cell lines among all PI3K/AKT/mTOR inhibitors tested. GSK458 has previ- ously been reported to be an efficient PI3K/mTOR dual inhib- itor with an inhibitory constant (Ki) ranging from 0.01 to 0.3 nM by Steven and colleagues in 2010 [34]. A previous phase I clinical trial study showed that GSK458 has few adverse ef- fects following oral administration and can produce durable objective responses (OR) in multiple tumor types (sarcoma, kidney, breast, endometrial, oropharyngeal, and bladder can- cer) with two patients having ongoing responses lasting more than 4 years [35]. However, to date there are no thorough reports on antitumor activity of GSK458 in ovarian cancer. Here, we show that GSK458 may serve as a potent candidate for the development of targeted therapies against ovarian can- cer. We also provide comparative data on the efficacy of var- ious PI3K/AKT/mTOR inhibitors for further preclinical or clinical studies on ovarian cancer. Since the safety and anti- tumor activity of GSK458 has already been reported in a phase I clinical trial study [35], we believe it is a strong can- didate for future preclinical and/or clinical studies.

⦁ Materials and methods

⦁ KEGG pathway enrichment analysis

Ovarian tumor messenger RNA sequencing data from The Cancer Genome Atlas (TCGA) were downloaded (https:// HiSeqV2_exon.gz) and analyzed. The data were divided into two groups (“early” and “advanced”) according to clinical stages determined by the International Federation of Gynecology and Obstetrics (FIGO) stage criteria. The early group includes patients at stage II (21 patients), and the ad- vanced group includes patients at stage III (233 patients) and IV (38 patients). KEGG pathway enrichment analysis was performed using the DESeq (2012) R package with default parameters according to a previous study [36] to detect the most activated pathways in advanced ovarian tumors.

⦁ Inhibitors

In total 16 PI3K/AKT/mTOR inhibitors were acquired from Selleck (Houston, TX, USA). This inhibitory panel includes 5 PI3K inhibitors [XL-147 (#S7645), IPI-145 (#S7028), BYL719 (#S2814), CAL-101 (#S2226), and GDC-0491 (#S1065)], 3 AKT inhibitors [GSK2141795 (#S7492), GSK2110183 (#S7521) and Triciribine (#S1117)], 4 mTOR
inhibitors [Rapamycin (#S1039), CCI-779 (#S1044), RAD001 (#S1120) and FK506 (#S5003)], 1 PI3K/AKT in-
hibitor [Miltefosine (#S3056)] and 3 PI3K/mTOR dual inhib- itors [GSK458 (#S2658), XL765 (#S7646) and SAR245409
(#S1523)]. Paclitaxel (#T7191) was purchased from Sigma- Aldrich (St. Louis, MO, USA). Cisplatin (#HY-17394) was purchased from MedChemExpress (MCE, NJ, USA). All compounds were dissolved in DMSO at a concentration of 10 mM and stored at -80 before use.

⦁ Ovarian cancer cell lines and primary ovarian tumor cell lines

The cell lines used in this study cover the 4 main subtypes of epithelial ovarian cancers: serous carcinoma (OVCAR3, OVCAR8), endometrioid carcinoma (TOV112D, COV362), mucinous carcinoma (MCAS) and clear cell carcinoma (ES2, OVISE, OVTOKO). Additionally, several widely used epithe- lial ovarian cancer cells of unclear subtype (A2780, A2780CP, SKOV3, SKOV3TR and SKOV3IP) or with a mixed subtypes (IGROV-1 and HO8910PM), were included [37, 38]. A nor- mal ovarian epithelial cell line HOSEPIC was used as control. All epithelial cell lines were cultured in complete RPMI-1640 culture medium (GIBCO, NE, USA) containing 10% fetal bovine serum (FBS; GIBCO), 100 U/ml penicillin (GIBCO) and 100 µg/ml streptomycin (GIBCO).
Primary ovarian tumor cells were cultured as previously described [33]. Briefly, freshly isolated tumor tissues from 4 patients (GFY005, CZ001, CZ006 and CZ008) undergoing surgery or from a third generation patient (GFY004)-derived xenograft (PDX) inoculated in the front flank of BALB/c nude mice, were cut into 1–2 mm diameter pieces. Then tissues were digested using a Tumor Dissociation Kit (130-095-929,
Miltenyl, Teterow, Germany) in a water bath at 37 for 45 min. Single cell suspensions were centrifugated and washed twice in RPMI-1640 medium at 150 x g for 5 min. Primary ovarian tumor cells were cultured in RPMI-1640 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin and 20% FBS for 10 to 20 generations before experiments. The characteristics of the patients included are listed in supplemen- tal table S1. Specifically, GFY004, GFY005 and CZ006 are high-grade serous carcinomas, CZ001 a is clear cell carcino- ma and CZ008 is a poorly differentiated carcinoma.

⦁ Cell proliferation assay

Cells (2 × 104/ml) in a total volume of 100 µl were seeded into 96 well plates and cultured overnight before treatment with various inhibitors. Inhibitors were added at 3-fold serial dilu- tions from 33.33 to 0.015 µM. After culturing for an addition- al 72 h, cell viability was assayed by a 3- (4,5-dimethylthiazol- 2-yl)- 5- (3-carboxymethoxyphenyl)- 2- (4- sulfophenyl)- 2H- tetrazolium (MTS) assay using a Cell Proliferation Assay kit (#G5430, Promega, Wisconsin, USA) according to the man- ual. IC50 was calculated using untreated cells as controls. All inhibitors were added at the same time to the same cell lines.

⦁ Cell migration assay

Cell migration assays were performed as described before [33]. Briefly, to compare the inhibitory effects on migration of PI3K/AKT/mTOR inhibitors, a total of 1 × 105 cells in 200 µl RPMI-1640 medium containing different inhibitors, with- out FBS, were seeded into 8 µm pore-transwell polycarbon- ate membrane filters (Costar Group, DC, USA) followed by insertion into 24 well culture plates. All inhibitors were used at a concentration of 1 µM, except for GSK458 and paclitaxel which were used at a concentration of 0.1 µM. To determine whether GSK458 inhibited cell migration in a dose dependent manner, 5 × 104 cells per transwell were treated with a 10-fold serial dilution of GSK458 from 1 to 0.001 µM. A volume of 700 µl RPMI-1640 medium containing 10% FBS was added into each well of the 24 well plates. After culturing for another 18 h, cells were fixed with 4% paraformaldehyde and stained with 2% crystal violet. Non- migrating cells on the upper surface of the chamber were scraped off with cotton swabs. Migrated cells at the bottom of the membrane were photographed at five random fields under a microscope with a magnification of 200x and were counted. Migration inhibition (percentage of control) was de- fined as the difference between migrating cells in the untreated control compared to inhibitor treatment divided by the control value. All inhibitors were added at the same time to the same cell lines.

⦁ Cell cycle assay

SKOV3 cells (1 × 105/ml) were seeded into 6 well plates in 2 ml complete culture medium and cultured overnight. All inhibitors were used at a concentration of 1 µM, except GSK458 and paclitaxel which were used at a concentration of 0.1 µM. The cells were cultured for an additional 24 h, harvested and fixed with 70% ethanol at 4 for another 24 h. The fixed cells were stained with 50 µg/ml propidium iodide (PI) containing 400 U/ml RNase A for 30 min at room tem- perature in the dark. Cell cycle distributions were determined using a FACSCalibur flow cytometer (BD Biosciences, CA., USA) within 2 h after staining and analyzed by FlowJo soft- ware (FlowJo LLC, OR, USA).

⦁ Xenograft nude mice model

Cancer cells were inoculated intraperitoneally (IP) into mice to mimic metastasis of ovarian cancers in the abdomen, as shown in previous studies [33, 39–42]. Five to six weeks old female BALB/c athymic nude mice were inoculated IP with 1 × 106 SKOV3 cells or 5 × 106 GFY004 cells in a total volume of 100 µl phosphate-buffered saline (PBS) containing 25% matrigel matrix (#356237, ThermoFisher Scientific). After seven days, mice were treated intraperitoneally with 2 mg/kg GSK458 or paclitaxel every 3 days for 4 consecutive weeks. When mice were sacrificed, all tumor nodes from abdominal tissues or organs, such as small intestinal, colon, mesenterium, liver, stomach and muscular tissues of abdominal cavity were surgi- cally removed, photographed, counted and weighed.

⦁ Western blot analysis

For the detection of intracellular levels of phosphorylated AKT (pAKT), cells or tissues were lysed using RIPA buffer contain- ing protease and phosphatase inhibitors (Beyotime, Jiangsu, China). Protein concentrations were measured using a BCA protein assay kit (ThermoFisher Scientific) for normalization. Twenty µg total protein from A2780, A2780CP, IGROV-1, SKOV3, SKOV3TR, SKOV3IP, MCAS, HO8910PM,
CZ001, CZ006, GFY004 and GFY005 cells, 60 µg total pro- tein from ES2, OVISE, TOV112D, COV362, OVTOKO,
OVCAR3, OVCAR8 and HOSEPIC cells, and 100 µg total protein from ovarian cancer tissues or normal ovarian epithelial tissues were loaded into 10% SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes.
The following antibodies were used to detect protein ex- pression or phosphorylation: antibodies directed against AKT (#8272), pAKT (Ser473) (#8271) and mTOR (#2983), pur-
chased from Cell Signaling Technology (CST, Danvers, MA, USA). An anti-p-mTOR (Ser2448) antibody (#AF3308) was purchased from Affinity Biosciences (Affinity, Cincinnati, OH, USA). A goat anti-rabbit (#926-32211, LI-COR) IRDye
800CW labeled secondary antibody was used for detection by Image Studio Version 5.2 on an Odyssey CLx infrared imag- ing system (LI-COR).

⦁ Immunohistochemistry

Tumors derived from nude mice were fixed with 4% parafor- maldehyde, embedded in paraffin and cut into 5 µm slices. Expression of and/or phoshporylation of AKT, mTOR and Ki67 was detected by immunohistochemistry using antibodies directed against AKT (#8272, CST), pAKT(Ser473) (#8271, CST), Ki67 (#GB13030-2, Servicebio, Wuhan, China), mTOR (#2983, CST) and p-mTOR(Ser2448) (#AF3308,

⦁ Statistical analysis

All experiments were performed at least in triplicate. Data are presented as mean ± standard deviation (SD). Statistical anal- yses were performed using GraphPad Prism 5.0 software (GraphPad Software Inc., ca., USA). Significant differences were determined by two-tailed Student’s t-test for Gaussian distribution data and by Mann-Whitney nonparametric test for non-Gaussian distribution data. P < 0.05 was considered sta- tistically significant.

⦁ Results

⦁ The PI3K/AKT signaling pathway is activated in advanced ovarian cancers

Signaling molecules that play critical roles in cancer progres- sion and/or recurrence possess a high potential as therapeutic targets. To determine the most important pathways activated in ovarian cancer, specifically in tumors from high grade pa- tients, RNA sequencing data of ovarian cancers from TCGA were mined using KEGG pathway enrichment. These RNA sequencing data were divided into two groups based on the FIGO stage criteria of patients. The early stage group included patients at stage II, while the advanced stage group included patients at stage III and IV. KEGG pathway enrichment anal- ysis identified the PI3K/AKT pathway as one of the most activated signaling pathways in advanced stage ovarian cancer patients (Fig. 1a).
To validate increased activation of the PI3K/AKT pathway in ovarian cancers, AKT phosphorylation was detected by Western blotting. Phosphorylation of AKT at Ser473 corre- lates well with that at Thr308, while the phosphorylation ex- tent of AKT at Ser473 is much stronger than that at Thr308. Also, mTORC2 activates AKT by phosphorylation at Ser473. So, in this study we probed phosphorylation of AKTat Ser473 as a readout of AKT activity. AKT phosphorylation (Ser473)

was assessed in 15 ovarian cancer cell lines (A2780, A2780CP, IGROV-1, SKOV3, SKOV3TR, SKOV3IP, MCAS, HO8910PM, ES2, OVISE, TOV112D, COV362,
OVTOKO, OVCAR3 and OVCAR8). We found that AKT phosphorylation could be detected in all cell lines tested (Fig. 1b). We also found that AKT phosphorylation was rela- tively weaker in the normal ovarian epithelial cell line HOSEPIC, compared to the 15 ovarian cancer cell lines. In the primary samples, AKT phosphorylation was higher in ovarian cancer tissues than normal ovarian epithelial tissues from the same patients (Fig. 1c). Detail characteristics of all
ƒFig. 1 The PI3K/AKT signaling pathway is highly activated in advanced ovarian cancers and ovarian cancer cell lines. a, KEGG pathway
enrichment analysis comparing ovarian tumors at stage II to stage III and IV using TCGA mRNA sequencing data. The bar graph shows the statistical significance of the enriched pathways. The arrow points to the PI3K/AKT signaling pathway as one of the highest activated signaling pathways in stage III and IV ovarian tumors compared to stage II tumors. b, AKT expression and phosphorylation (Ser473) levels in 15 ovarian cancer cell lines (A2780, A2780CP, IGROV-1, SKOV3, SKOV3TR, SKOV3IP, MCAS, HO8910PM, ES2, OVISE, TOV112D, COV362,
OVTOKO, OVCAR3 and OVCAR8) and a normal ovarian epithelial cell line (HOSEPIC) detected by Western blotting. Left panel, 20 µg total protein of each cell line was analyzed. Right panel, 60 µg total protein of each cell line was analyzed. The lower panel shows the relative activity of AKT compared to the expression of GAPDH, which was calculated by the ratio of the density of pAKT bands to the corresponding GAPDH bands. The density of bands was measured using Image Studio Version 5.2 on an Odyssey CLx infrared imaging system (LI-COR). Statistical analyses were carried out between each cell line and HOSEPIC cells. c, AKT phosphorylation (Ser473) detected in ovarian tumors and normal ovarian tissues from the same patients. One hundred µg total protein of each tissue was analyzed. ca.: carcinoma, CN: normal ovarian tissue control. The lower panel shows the relative activity of pAKT calculated as described above. The pAKT activity in normal control tissues was set at 1.0. d, AKT phosphorylation (Ser473) was universally detectable in primary ovarian cancer cell lines. SKOV3 and HO8910PM served as controls. Twenty µg total protein of each cell line was analyzed. Data are presented as mean ±
SD. * p < 0.05; ** p < 0.01; *** p < 0.001

the patients with epithelial ovarian cancer are shown in sup- plemental Table S1. AKT phosphorylation was also observed in all primary ovarian cancer cell lines tested (Fig. 1d).

⦁ PI3K/AKT/mTOR inhibitors cause SKOV3 cell cycle arrest at the G0/G1 phase

Classical PI3K/AKT/mTOR inhibitors are known to induce cell cycle arrest at the G0/G1 phase. We assessed their effects on cell cycle distribution in SKOV3 cells using flow cytome- try. We indeed found that almost all PI3K/AKT/mTOR inhib- itors tested caused SKOV3 cell cycle arrest at the G0/G1 phase, while paclitaxel caused SKOV3 cell cycle arrest at G2/M phase (Fig. 2a and b). Treatment with GSK458, XL- 765, Rapamycin, CCI-779, RAD001, GSK2110183,
GSK2141795, BYL719 or GDC-0941 led to higher percent- ages (70% − 90%) of G0/G1 phase cells than Miltefosine, FK506, XL147, IPI-145, CAL-101, SAR245409 or
Triciribine (60% − 70%).

⦁ GSK458 inhibits ovarian cancer cell proliferation and migration

The effect of PI3K/AKT/mTOR inhibitors and paclitaxel on inhibiting the proliferation of ovarian cancer cells was de- tected using an MTS assay. We found that most inhibitors tested had little to no inhibitory effect on the proliferation of

Fig. 2 GSK458 potently inhibits the proliferation and migration of ovarian cancer cell lines and primary ovarian tumor cell lines. a, SKOV3 cells were treated with 1 µM of various inhibitors, except GSK458 and Paclitaxel (0.1 µM), for 24 h. Untreated cells were used as negative controls. Cell cycle distribution was detected by PI staining on a FACSCalibur (BD Biosciences) and analyzed by flowjo software.
Representative cell cycle distribution images of untreated control, paclitaxel- or GSK458- treated cells are shown. b, Statistical cell cycle distribution analysis. G0/G1: G0/G1 phase; S: S phase; G2/M: G2/M phase. c, d and e, IC50 values of PI3K/AKT/ mTOR inhibitors and paclitaxel detected using a MTS assay. The cells were treated with various inhibitors at concentrations from
0.015 to 33.33 µM. IC50 values lower than 0.015 µM or higher than 33.33 µM were set at 0.015 and 33.33 µM, respectively. f, g and h, Migration inhibition by PI3K/AKT/mTOR inhibitors and paclitaxel on cell lines measured by transwell assay. Differences in inhibitory efficacies between GSK458 and paclitaxel on cell proliferation (e) and migration (h) were statistically analyzed. Statistic data are presented as mean ± SD. * p < 0.05; **,
p < 0.01; *** p < 0.001

ovarian cancer cells (Fig. 2c and d). The IC50 values of 10 inhibitors (Miltefosine, Rapamycin, CCI-779, RAD001, FK506, XL147, IPI-145, CAL-101, SAR245409 and
Triciribine) were higher than the maximal detectable concen- tration of 33.33 µM in several of the cell lines tested (Fig. 2d), which means they have a low efficiency. GDC-0941 had an IC50 less than 10 µM in all cell lines tested, except the MCAS and GFY004 cell lines (Fig. 2c). Interestingly, we found that the IC50 values of 5 inhibitors, GSK458, XL- 765, GSK2110183, GSK2141795 and BYL719, were all
lower than 33.33 µM in all cell lines tested (Fig. 2c). Among all inhibitors tested across the different cell lines, GSK458 had the lowest IC50 value (0.01 to 1 µM), which rivals the front-line drug paclitaxel (Fig. 2e). The IC50 data of Fig. 2c, d and e are shown in supplemental Table S2.
In vitro cell migration assays can assess the invasive and metastatic capacities of primary tumors. We tested the effects of the PI3K/AKT/mTOR inhibitors on ovarian cancer cell migration in 3 ovarian cancer cell lines (SKOV3, ES2 and OVCAR8) and 3 primary ovarian tumor cell lines (CZ006, GFY004 and GFY005) with known high migratory capaci- ties. The migration inhibition results are shown in Fig. 2f, g and h, and the corresponding mathematical data are shown in supplemental Table S3. We found that the migration inhibitory capacities of 8 inhibitors (Miltefosine, FK506, XL147, GSK2110183, GSK2141795, BYL719, CAL-101, and
SAR245409) were all lower than 50% in all cell lines tested. The migration inhibitory capacities of 4 inhibitors (Rapamycin, CCI-779, RAD001 and Triciribine) were higher than 50% in only one of these six cell lines. The migration

inhibitory capacities of IPI-145 and GDC0941 in two of these six cell lines were higher than 50%. Together, these data mean that these 14 inhibitors had little or no effect on the inhibition of ovarian cancer cell migration. The migration inhibitory capacities of XL-765 and paclitaxel in five of these six cell lines were higher than 50%. Importantly, we found that the migration inhibitory capacity of candidate drug GSK458 in all detected cell lines was higher than 50% with a range from 70– 90%. Statistical analysis revealed that the migration inhibitory capacity of GSK458 on all cell lines tested was significantly higher than that of paclitaxel (Fig. 2h). Together, we found that GSK458 exhibited the highest inhibitory activity against ovarian cancer cell proliferation and migration among all PI3K/AKT/mTOR inhibitors tested. The anti-proliferative and anti-migrative activities of GSK458 were comparable to or even higher than those of paclitaxel.
To investigate whether GSK458 can inhibit ovarian cancer cell migration in a dose dependent manner, SKOV3 or GFY004 cells were treated with GSK458 at increasing doses. We found that GSK458 strongly inhibited the migration of
both SKOV3 (Fig. 3a, b and c) and GFY004 (Fig. 3d, e and f) cells in a dose dependent manner. The 50% inhibitory con- centrations of GSK458 in SKOV3 (Fig. 3c) and GFY004 (Fig. 3f) cells were lower than 0.001 µM. Most importantly, only a few scattered cells could migrate through the transwell mem- brane after treatment at higher GSK458 concentrations (0.1 or 1 µM; Fig. 3a and d).

⦁ GSK458 inhibits ovarian cancer growth and metastasis in nude mice

Next, we decided to test the efficacy of GSK458 in vivo using a mouse model that may better simulate the heterogeneity and genetic complexity of tumors as demonstrated by our previous work [33]. Five to six weeks old female BALB/c athymic nude mice were inoculated intraperitoneally with SKOV3 cells or patient-derived GFY004 cells (PDCX) and treated with 2 mg/kg GSK458 or the front line drug paclitaxel. After the mice were sacrificed, all tumor nodes were surgically removed. We found that both GSK458 and paclitaxel strongly inhibited

Fig. 3 GSK458 strongly inhibits ovarian cancer cell migration. a and d, SKOV3 and GFY004 cells were treated with increasing concentrations of GSK458 as indicated. Migrated cells were detected using a transwell migration assay. b and e, Statistical data of migrated SKOV3 and GFY004 cells, respectively. c and f, Migration inhibition by GSK458 of SKOV3 and GFY004 cells at the indicated concentrations, respectively.
Images were taken at a 200x magnification. Error bar: mean ± SD. *** p < 0.001

tumor growth and metastasis in mice inoculated with GFY004 (Fig. 4a, b and c) or SKOV3 (Fig. 4e, f and g) cells. Differences between the GSK458 and paclitaxel treated groups were not significant, suggesting that the anti-growth and -invasion effects of GSK458 are at least comparable to that of paclitaxel (Fig. 4a, b and c). Both GSK458 and paclitaxel did not affect the body weights of the mice (Fig. 4d and h).

⦁ GSK458 inhibits the PI3K/AKT/mTOR signaling pathway

The effect of GSK458 on the PI3K/AKT/mTOR signaling pathway was assessed by Western blotting and immunohisto- chemistry. We found that GSK458 efficiently blocked AKT and mTOR phosphorylation in SKOV3 cells in vitro, even at a low concentration (0.1 µM; Fig. 5a). Tumors derived from GSK458 treated nude mice also showed striking decreases in AKT and mTOR phosphorylation (Fig. 5b). In addition, we found that expression of the tumor cell proliferation mark- er Ki67 was inhibited by GSK458 in tumors derived from nude mice inoculated with SKOV3 cells (Fig. 5b). These data indicate that the PI3K/AKT/mTOR signaling pathway is inhibited by GSK458. The inhibitory effect of GSK458 on the PI3K/AKT/mTOR signaling pathway leading to tumori- genesis inhibition is depicted in Fig. 5c.

⦁ Discussion

Ovarian cancer is the fifth leading cause of death among pa- tients with gynecological malignancies [2]. Targeted therapy may be one of the most effective strategies to improve the overall survival (OS) of ovarian cancer patients [43]. This requires a deeper understanding of the molecular mechanisms underlying ovarian cancer development. In this study, we use KEGG pathway enrichment analysis on RNA sequencing data from TCGA and found that the PI3K/AKT pathway was one of the most elevated signaling pathways in advanced ovarian tumors compared to earlier stages. The PI3K/AKT pathway has suggested to be an attractive target for ovarian cancer therapy. The TCGA consortium has reported that the KRAS/ PI3K axis is activated in 45% of the cases (316 patients), and that the PI3K/AKT/mTOR pathway is either mutated or amplified in 34% of the cases [14, 44]. This suggests that the PI3K/AKT/mTOR pathway may play a critical role in ovarian cancer development and progression [44]. At present, dozens of PI3K/AKT/mTOR inhibitors have entered clinical trials for ovarian cancer therapy and shown some benefits. It is, however, difficult to directly compare the efficacies of the individual candidate inhibitors reported due to lack of exper- imental standardization. In our current study we used stan- dardized methodologies to identify potent PI3K/AKT/ mTOR inhibitors that can be used for ovarian cancer therapy.

Fig. 4 GSK458 strongly inhibits ovarian cancer tumorigenesis and metastasis in vivo. BALB/c athymic nude mice were intraperitoneally inoculated with 5 × 106 GFY004 cells (a, b, c and d) or 1 × 106 SKOV3 cells (e, f, g and h). Seven days after inoculation, the mice were treated with 2 mg/kg GSK458 or paclitaxel every 3 days for 4 weeks. Other mice treated with the same volume of DMSO served as negative controls. a and e, All visible tumors from each mouse were isolated, placed into a 6-cm diameter cell culture dish and photographed. b and f, Tumor numbers from each nude mouse were calculated. c and g, Tumor weights from each nude mouse were recorded. d and h, Mouse body weights determined just before sacrifice. CON: DMSO controls. PAC: paclitaxel. Error bar: mean ± SD. ** p < 0.01; *** p < 0.001

We screened a panel of in total 16 drug candidates in parallel. Using both ovarian cancer cell lines and primary tumor cell lines, we observed a wide range of inhibitory efficacies across the inhibitors tested. The cell lines used in this study covered not only the 4 main subtypes of epithelial ovarian cancer (se- rous carcinoma, endometrioid carcinoma, mucinous carcino- ma and clear cell carcinoma), but also some widely used mod- el cell lines of unknown origin or mixed subtypes. BALB/c

Fig. 5 GSK458 inhibits the PI3K/AKT/mTOR signaling pathway. a, SKOV3 cells were treated with drugs for 24 h to assess their impact on the PI3K/AKT/mTOR signaling pathway. GSK458 at two different concentrations (1 and 0.1 µM) was compared to 0.1 µM paclitaxel (PAC 0.10) and 10 µM cisplatin (CIS 10). Cells treated with DMSO served as negative controls. Western blot analysis was used to assess AKT and mTOR expression (AKT, mTOR) and phosphorylation (pAKT, pmTOR). GAPDH was used as control. Cells treated with
DMSO served as negative controls. b, Expression and/or phosphorylation of Ki67, AKT and mTOR in tumors derived from nude mice treated with 2 mg/kg GSK458 for every 3 days for 4 weeks detected by immunohistochemistry. Tumors derived from nude mice treated with the same volume of DMSO served as negative controls. c, Diagram illustrating the inhibitory mechanism of GSK458 on PI3K/AKT/mTOR signal transduction

nude mice were used to assess the antitumor efficacy of high potent candidates in vivo. The mice were injected intraperito- neally with ovarian cancer cells to mimic abdominal metasta- sis of ovarian cancer in vivo. Although this model cannot differentiate primary tumor cell dissemination from metastasis nodes, it is widely accepted and used in ovarian cancer metas- tasis studies [33, 39–42]. Surprisingly, although almost all inhibitors caused to some extent G0/G1 phase cell cycle arrest, more than half of the tested inhibitors showed minimal effects on inhibition of ovarian cancer cell proliferation and/or migra- tion, including two lymphoma drugs (CAL-101 and IPI-145) approved by the U.S. Food and Drug Administration (FDA) [45]. Only three candidates (GSK458, XL765 and GDC- 0941) exhibited high inhibitory effects both on proliferation and migration. We found that GSK458 had the highest anti- proliferative and -migrative activity among the candidate PI3K/AKT/mTOR inhibitors tested in parallel. It has amply been reported that PI3K activation induces phosphorylation and activation of AKT and mTORC1, which in turn activates S6K followed by induction of Cyclin D. Cyclin D binds CDK4/6 to promote RB phosphorylation and release from the E2F transcription factor, which drives expression of genes for cell cycle progression and cell mobility [46, 47]. A previ- ous study also showed that GSK458 can dephosphorylate and inactivate AKT and S6K to induce G0/G1 cell cycle phase arrest [48]. Also, it has been found that mTORC1 can inhibit the activity of mTORC2 followed by inactivation of SGK and
PKC, which in turn induces cell migration [49]. Our in vivo study supported our in vitro findings that GSK458 can inhibit ovarian cancer tumorigenesis and metastasis. Most important- ly, we found that the anti-proliferative and -migrative effects of GSK458 in vitro and in vivo were comparable, and in some instances even better than those of paclitaxel. Consistent with our results, a phase I clinical trial reported that GSK458 treat- ment led to durable objective responses (OR) in multiple can- cer types, including soft tissue, kidney, breast, endometrial, oropharyngeal and bladder cancer, with two patients develop- ing tumor responses that lasted more than 4 years [35]. Combination treatment of GSK458 with another therapeutic agent, such as the MEK inhibitor GSK1120212, has also been found to enhance the antitumor activity [50].
It has been reported that mTORC1/2 inhibition may pro- vide added benefit in reversing platinum resistance compared to mTORC1 inhibition alone, promoting the survival of plat- inum resistant ovarian cancer patients [26]. The PI3K/mTOR dual inhibitor GDC-0941 has been found to strongly inhibit ovarian cancer cell proliferation, except in mucinous MCAS cells, which contain an activating mutation in the PIK3CA gene [28]. GSK458, as a pan PI3K/mTOR dual inhibitor, has been found to exhibit a high antitumor activity in all ovar- ian cancer cell lines tested, including MCAS. In accordance with our results, Munster and colleagues have reported that patient responses to GSK458 were not correlated with PIK3CA mutations [35]. Since only six ovarian cancer

patients were included in that study, the relationship between the antitumor effects of GSK458 on ovarian cancer cells and the presence of PI3KCA mutations requires further investiga- tion [35]. GSK458 has been shown to be a substrate for P- glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), which decrease the distribution and anti-tumor effects of GSK458 in tumors [51]. Here, we included three AKT- specific inhibitors (GSK2141795, GSK2110183 and Triciribine) and four mTOR-specific inhibitors (Rapamycin, CCI-779, RAD001 and FK506). We found that all AKT- and mTOR-specific inhibitors tested had lower activities than GSK458 in inhibiting ovarian cancer cell proliferation and migration. As GSK458 is a PI3K/mTOR dual inhibitor, the role of synergistic inactivation of both AKT and mTOR should be considered as a critical biomarker for evaluating the antitumor effects of GSK458 in clinical trials. Previously, it has been shown that miRNAs can regulate sev- eral signaling pathways, including the TP53, TGF-β, PI3K/AKT/mTOR, MAPK and HIF-1 pathways and, thereby, impact the prognosis of ovarian cancer patients [52]. Kanlikilicer and colleagues reported that miR-6126 can re- duce the phosphorylation of PI3K and AKT in high grade serous ovarian cancer and is associated with a longer survival [53]. It has also been reported that mTOR is a target of miR- 100, miR-199a and miR-497, which may affect the anti-tumor efficacy of mTOR inhibitors [54]. Based on these findings, additional biomarkers for the anti-tumor efficacy of PI3K/AKT/mTOR inhibitors, such as GSK458, may improve the clinical outcome. Although there are no clinical studies of the anti-tumor effects of GSK458 in ovarian cancer, the re- sponses to GSK458 observed in other advanced solid tumors in a previous phase I clinical trial [35] and our preclinical study warrant further preclinical and/or clinical studies focus- ing on the effect of GSK458 on ovarian cancer.

⦁ Conclusions

In summary, our data indicate that the PI3K/AKT/mTOR pathway is strongly activated in ovarian tumors from high stage patients. Among all the tested PI3K/AKT/mTOR inhib- itors, GSK458 exhibited the highest antitumor activity, both in vitro and in vivo. This antitumor effect of GSK458 was comparable to that of paclitaxel. Our study strongly suggests that GSK458 may be an attractive candidate for further studies aimed at therapeutically targeting ovarian cancer.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81702563 to Y.J.X., and 81472445 and 81672587 to R.Z.), the Postdoctoral Science Foundation of China (Grant No. 2019M652850 to Y.J.X.) and the Scientific and Technological Innovation Act Program of the Shanghai Science and Technology Commission (Grant No. 4411973100 to R.Z.).
Compliance with ethical standards Written informed con- sent was obtained from all individual participants according to the Declaration of Helsinki. This study was carried out in accordance with the recommendations of, and approval by the Ethical Committee of Fengxian District Central Hospital. All animal experimental procedures described in this study were performed according to the Guidelines for the care and use of animals established by the Laboratory Animal Ethical Board and approved by the Animal Care and Use committee of the East China Normal University School of Life Sciences.

Conflict of interest The authors declare that they have no competing interests.


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