Identification of cell cycle dependent methotrexate resistance in placenta site trophoblastic tumors

Background The purpose is to study the mechanism of chemotherapy resistance in Placental site trophoblastic tumor(PSTT). Methods We established PSTT cell lines by primary culture of a surgically resected PSTT tissues and identified the expression of immune-phenotype markers(HLA-G, β-catenin, CD146, Muc4, hPL, hCG) by immunofluorescence. We measured the IC50 value of methotrexate(MTX), etoposide(VP-16), actinomycin-D(Act-D), cisplatin(DDP), fluorouracil(5-FU) and paclitaxel(TAX) in PSTTs and used a special Mini patient-derived xenograft (Mini PDX) model to evaluate effectiveness of these drugs in vivo . Given that MTX is a cell cycle-dependent chemotherapeutic, we analyzed cell cycle characteristics of PSTT and choriocarcinoma cell lines by flow cytometry and then analyzed RNA profiles and WGS data of the PSTT cell lines to identify the potential mechanism. Results We identified the expression of HLA-G, β-catenin, CD146, hPL and hCG in PSTT cell lines. The IC50 value of MTX was 4.922 mg/ml in PSTT-1, 4.525 mg/ml in PSTT-2, 5.117 mg/ml in PSTT-3, 0.0166 µg/ml in JEG-3 cells (p0.001), and 0.01 µg/ml in JAR cells (p0.001), with nearly 50,000-fold increase in PSTTs than in choriocarcinoma, indicating that PSTTs are resistant to MTX in vitro. The Mini PDX model revealed that PSTTs are also resistant to MTX in vivo . Cell cycle analysis showed dysregulation of G1/S transition and cell cycle arrest in PSTT cell lines. RNA sequencing profile also identified cell cycle-associated genes which were differentially expressed in PSTT cells than in choriocarcinoma cell. Conclusions We found PSTTs compared to choriocarcinoma.


Mini PDX drug sensitivity assays:
BALB/c-nude mice(5-6 weeks) bearing Mini-PDX capsules were treated with drugs as detailed in Tables 1 for 7 days. Thereafter, the implanted capsules were removed, and tumor cell proliferation was evaluated using CellTiter Glo Luminescent Cell Viability Assay kit (G7571, Promega, Madison, WI, US) as instructed by the manufacturer. Luminescence was measured in terms of relative luminance unit (RFU) using a spectrophotometer (SpectraMax M3, Molecular Devices, Sunnyvale, CA, US). Tumor cell growth inhibition (TCGI) (%) was calculated using the following formula: TCGI%=1-(Mean RLU of the treatment group on day 7-Mean RLU on day 0)/(Mean RLU of the vehicle group on day 7-Mean RLU on day 0)×100% Each experiment was done in sextuplicate and mean values were reported. A positive drug response was considered present if TCGI was≥45% (P<0.05), and a negative drug response was considered if TCGI was 45% (P<0.05).

Cell cycle analysis:
After the indicated treatments, the cells were resuspended in PBS and fixed in 70% ethanol-PBS for 30 min at 4 °C. Fixed cells were washed with PBS and treated with RNase at 37 °C for 30 min. Finally, the cells were stained with propidium iodide (PI) for 30 min at 37 °C. Samples were analyzed by flow cytometry using a FACSort cytometer (Becton-Dickinson, Franklin Lakes, NJ) and Cell Quest and ModFit software.

RNA-expression data retrieval and analysis:
Total RNA was extracted from cells using an RNA isolation kit (QIAGEN, Germany). GO enrichment and KEGG pathway analysis were implemented to discover pathways/processes in which the DEGs participated. The enriched functions of the genes were analyzed according to the GO terms of biological process (BP), cellular component (CC), and molecular function (MF). KEGG (http://www.genome.jp/KEGG/) is an online database that contains information on genomes, enzymatic pathways and biochemical. The DAVID (https://david.ncifcrf.gov/) was used to annotate and analyze the associated GO terms and KEGG pathways of the DEGs, with p<0.05 indicating significance.

WES and Data processing:
Genomic DNA was extracted from three PSTT cell lines with the DNeasy Kit (QIAGEN, Germany). Nextgeneration sequencing was conducted with the Illumina HiSeq X-TEN platform at CloudHealth Genomics. Reads were aligned to the human genome reference assembly (UCSC Genome Browser hg19) with the Burrows-Wheeler Aligner. Single-nucleotide variants (SNVs) and indels were called and analyzed by the Genome Analysis Toolkit. The SNVs with read depths less than 43 were filtered out.

Result 1Establishment and identification of three PSTT cell lines
We established three PSTT cell lines derived from primary culture of surgically resected PSTT tissues from three PSTT patients. Immunofluorescence imaging showed that PSTT-1,2,3 cells all expressed HLA-G, β-catenin, CD146, Muc4, hPL and hCG, which are typical molecular markers of intermediate  Table 1]. Gene locus similarity between the PSTT cell lines and tumor tissues was as high as 100%(values greater than or equal to 80% are considered to indicate homologous samples).
Moreover, the karyotype of the PSTT cell line was 46,XX, the same as the normal female karyotype [ fig. 1C].

Result 2PSTT cell lines are resistant to MTX in vitro
The preferred first-line therapy for PSTT is EP/EMA or EMA/EP. Therefore, we used the established cell lines to study chemosensitivity of the drugs in the EMA/EP regimen, including MTX, etoposide(VP-16), actinomycin D(Act-D), and cisplatin(DDP). In addition, we studied paclitaxel(TAX) from the TP/TE regimen and fluorouracil(5-FU) from the FA regimen. As shown in Fig. 2A, MTX had a significantly higher IC50 value in three PSTT cell lines (4.922 mg/ml in PSTT-1, 4.525 mg/ml in PSTT-2, 5.117 mg/ml in PSTT-3) than in the choriocarcinoma JEG-3 (0.0166 μg/ml) and JAR cells (0.01 μg/ml). PSTT cell lines showed resistance to MTX, with a 50,000-fold increase in the IC50 value compared to that in choriocarcinoma cells. PSTT cell lines exposed to VP- 16 fig. 2F] showed no significant difference in IC50 value compared to JAR and JEG-3 cells. In summary, PSTT cell lines show greater resistance to MTX than to six other common chemotherapy drugs. Therefore, we decided to focus on MTX resistance in PSTT cell lines.

Result 4G1 phrase arrest and G1/S-phrase transition was dysregulated in PSTT cell lines
Considering that MTX, a folate antagonist, is traditionally regarded as a proliferation inhibitor that blocks the S phase of the cell cycle (15) , we analyzed the cell cycle characteristics of the three PSTT cell lines. We found that the proportion of cells in S phase in the PSTT cell lines[ fig. 4B] was significantly less than that in the JAR and JEG-3 cell lines[ fig. 4A], suggesting that G1 phase arrested and the G1/S transition was dysregulated in PSTT cell lines compared to choriocarcinoma cell lines[ fig.  4C]. As previously reported, cell cycle disorder is an important mechanism underlying the increased malignancy of tumor cells (22) , and alterations in the cell cycle have been considered responsible for drug resistance (23) . Therefore, we propose that loss of control of the G1/S transition may explain why PSTT is resistant to MTX in vivo and in vitro compared to MTX-sensitive choriocarcinoma cell lines.

Result 5Cell cycle associated DEGs were identified in PSTT cell lines
To determine whether loss of control of the G1/S transition underlies the MTX resistance in PSTT, we

Discussion
PSTT is a rare form of GTNs (2,3,24) and is less sensitive to conventional chemotherapy than choriocarcinoma. Hysterectomy is the most recognized treatment, which results in the loss of fertility (3-7) . Fertility-sparing therapy in PSTT patients is controversial because of the underlying chemoresistance. There are few reports on the basic mechanism of PSTT; the establishment of tumor cell lines plays an important role in basic tumor research, but there are currently no PSTT cell lines in authoritative cell banks. Thus far, there is only one report on the establishment of aPSTT cell line, IST-2 (25) . In contrast, we identified the expression of HLA-G, β-catenin, CD146, Muc4, hPL and hCG, which are more convicing markers of PSTT. The karyotype of IST-2 is 38-45, X,-X,-1,-2,-3,-6,-8,-22[cp11]/46,XX, and that of PSTT-1 is 46,XX, which mimics the normal female karyotype. These karyotype results are consistent with previous study, which revealed the presence of an X chromosome and the absence of Y chromosome was observed in 20 PSTT tissues (26) . Besides, we compared the homology between the PSTT-1 cell line and PSTT tissue by STR analysis, further validating of the PSTT-1 cell line. Shih's study (25) involved using IST-2 to explore the role of MAPK pathway in PSTT invasion and migration rather than the chemoresistant mechanism. Therefore, our study firstly investigated the chemotherapy mechanism in PSTT.
The preferred first-line therapy is EP/EMA or EMA/EP in NCCN Guidelines. Therefore, we studied the chemosensitivity of PSTT to drugs in the EMA/EP regimen, including MTX, VP-16, Act-D, and DDP. In addition, we studied TAX from the TP/TE regimen and 5-FU from the FA regimen, which are also common regimens. In vitro, we found that PSTT were quite resistant to MTX, with a nearly 50,000-fold increase in the IC50 value, suggesting the methotrexate resistance of PSTT cell lines. Then, we verified the EMAEP regime in vivo because of MTX. We found that PSTT were resistant to the four drugs, while JEG-3 was still sensitive to the four drugs. Regarding the different results in vivo and in vitro, we considered that the drug effect might differ in vivo and in vitro because of the microenvironment, resulting in differences in physiological drug metabolism. In addition, these four drugs are clinically used as a combination regimen, and drug interactions play a role in vivo; therefore, a single drug may not work in vivo, and the four drugs might have no inhibitory effect on the Mini-PDX model when given separately, resulting in inconsistent drug effects in vitro and in vivo.
The population of untreated JEG-3 cells generally increased almost 100-fold in 7 days, but that of PSTT cells increased no more than 2 fold, suggestive of the different growth rates of the two cell lines, which were also observed in vitro. In addition, PSTT cells could not form tumors by in situ or subcutaneous implantation, which may be also due to the slow growth.
MTX is the single most commonly used agent for the treatment of choriocarcinoma patients without metastases or with low risk of metastasis (12,13) , and approximately 30% of patients develop drugresistance for MTX monotherapy (14) . Without combining with other drugs to work, so it's relatively easy to study drug pharmacology and mechanism. All of the regimens for PSTT are combination therapies. The mechanisms of drug interactions are complicated, so it's difficult to perform drug effectiveness research, which may be why MTX-related studies in GTNs have mostly focused on drug resistance in choriocarcinoma. However, in our study, PSTT was least sensitive to MTX among the drugs in the commonly used combination chemotherapy regimen EMA/EP. JEG-3/MTX and JEG-3R cells have been used as resistance models (27,28) to identify mechanism for chemotherapeutic resistance to MTX in choriocarcinoma (29) . JEG-3R showed more than 20-fold resistance compared to JEG-3/MTX. However, in our study, PSTT cell lines showed almost 50,000-fold resistance compared to JAR and JEG-3 cells. Therefore, we consider MTX resistance to be a unique characteristic of PSTT. We suspect that if patients have a poor response to EMA/EP, it is likely due to the MTX. Therefore, through our research, we can provide new directions for future drug research on PSTT.
We firstly began to explore the mechanism of PSTT in the field of MTX resistance. According to previous researches, the most studied mechanism of MTX resistance is the anti-folate metabolic pathway (30,31) . MTX tightly binds to dihydrofolate reductase(DHFR), blocking the reduction of dihydrofolate to tetrahydrofolic acid. (32) . Resistance to MTX may develop as a result of elevated DHFR activity or defective transport of methotrexate into malignant cells (32) . However, research assessing whether DHFR expression is correlated with MTX resistance in JEG-3 showed that DHFR gene transcript level correlated with MTX drug resistance at only a certain phase; thus, DHFR expression levels would be of limited use as a biomarker for the prediction of MTX chemoresistance (33) . In addition to DHFR, methylenetetrahydrofolate reductase (MTHFR) is also an important rate-limiting enzyme in folate metabolism. Studies have demonstrated that the MTHFR 677TT genotype in molar tissue is associated with ineffective MTX treatment in Japanese low-risk GTN patients, but there was no verification of the mechanism relating MTHFR function to MTX sensitivity (34) . Besides, Lssecka et al. found that neither the genotype for the 677CNT SNP or the 1298ANC SNP predict the therapeutic outcomes of women treated with single agent MTX for GTN, indicating that no correlation between MTHFR polymorphisms in molar tissue and MTX drug resistance (35) . As a result, DHFR-and MTHFRassociated studies have not clearly delineated the role of folate metabolism in MTX resistance in choriocarcinoma and the role of expression level of DHFR and MTHFR polymorphisms in prediction of MTX resistance is controversial for now. Therefore, our study did not focus on the folate metabolism pathway.
We found that G1 phrase arrest and dysregulation of G1/S-phrase transition in PSTT cell lines.
Defective cell cycle events result in uncontrolled cell proliferation, which is considered as one of the hallmarks of cancer (22) . Oncogenic processes exhibit their greatest effects by targeting G1 phase progression (15) and alternations in cell cycle have also been considered responsible for drug resistance (23) . Many studies have verified that cell cycle disorders are related to MTX-resistant in some solid tumors (36,37) , therefore, we decided to focus our study of MTX-resistance on dysregulated cell cycle.
With the RNA sequencing profile, we found clusters of DEGs related to pathways involved in the cell cycle, suggesting that cell cycle-associated pathways are related to the MTX-resistance. Further verification of mechanism requires further study. We considered whether genetic mutation or epigenetic modification was responsible for these changes. We checked candidate genes of clusters

Authors' contributions:
The conception and design of study: Yu Kang and Congjian Xu; The acquisition of data: Jing Xu, Ling Zhang, Qiyu Liu;