Clinical significance of Gremlin 1 in cervical cancer and its effects on cancer stem cell maintenance

  • Authors:
    • Masakazu Sato
    • Kei Kawana
    • Asaha Fujimoto
    • Mitsuyo Yoshida
    • Hiroe Nakamura
    • Haruka Nishida
    • Tomoko Inoue
    • Ayumi Taguchi
    • Juri Takahashi
    • Katsuyuki Adachi
    • Kazunori Nagasaka
    • Yoko Matsumoto
    • Osamu Wada‑Hiraike
    • Katsutoshi Oda
    • Yutaka Osuga
    • Tomoyuki Fujii
  • View Affiliations

  • Published online on: October 30, 2015     https://doi.org/10.3892/or.2015.4367
  • Pages: 391-397
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Abstract

Gremlin 1 is one of the bone morphogenetic protein (BMP) antagonists and is also related to differentiation in combination with BMPs and is associated with various types of diseases. Gremlin 1 is overexpressed in various types of human cancers and has been reported to play a role in cervical cancer oncogenesis. However, there is no report concerning the relationship between Gremlin 1 and cervical cancer stem cells (CSCs). The objective of the present study was to identify the clinical significance of Gremlin 1 in cervical cancer and its effects on CSC‑like properties in vitro. Clinical samples were obtained. Gremlin 1 mRNA expression levels in the cervical cancer tissues were measured by RT‑qPCR and assessed for correlation with their clinical prognosis [overall survival (OS), progression-free survival (PFS)] and with other prognostic factors. In vitro, cervical cancer, CaSki cells, exposed to Gremlin 1 (1,000 ng/ml) for 24 h were evaluated for expression of undifferentiated‑cell markers (Nanog, Oct3/4, Sox2) by RT‑qPCR, the population of ALDH‑positive cells by flow cytometry and sphere‑forming ability on a ultra‑low attachment culture dish. Cervical cancer tissues from 104 patients were collected. A high mRNA expression level of Gremlin 1 was an independent poor prognostic factor of PFS but not of OS. A high mRNA expression level of Gremlin 1 was correlated with bulky (>4 cm) tumors. The Nanog mRNA expression level was significantly increased in the CaSki cells exposed to Gremlin 1 (P=0.0008) but not Oct3/4 and Sox2 mRNA expression levels. The population of ALDH‑positive cells in the Gremlin 1‑exposed cells was 1.41‑fold higher compared with the control (P=0.0184). Sphere‑forming ability was increased when 1,000 Gremlin 1‑exposed cells were seeded (P=0.0379). In cervical cancer, it is suggested that Gremlin 1 may have a role in clinical recurrence and maintaining CSC-like properties.

Introduction

The cancer stem cell (CSC) theory states that only a small population of cells within a tumor is tumorigenic and has the ability for self-renewal (1). CSCs are thought to be related to tumor recurrence and treatment resistance; even though we treat non-CSCs by surgery or chemotherapy, relapse or metastasis may result if CSCs should resist (2). This theory was first suggested in leukemia, and many studies investigating CSCs are now performed in various types of solid tumors such as glioblastoma, colon cancer, breast cancer and prostate cancer (37). Undifferentiated-cell (normal stem cell) markers such as Nanog, OCT3/4 and SOX2 are known to be expressed in a small population of cancer cells (817). Increasing evidence suggests that tumor tissues are not homogeneous but heterogeneous supporting the CSC hypothesis.

Gremlin 1 is one of the bone morphogenetic protein (BMP) antagonists and especially antagonizes with BMP2, BMP4 and BMP7 (18,19). BMPs are members of the transforming growth factor (TGF)-β superfamily and play an essential role during development and differentiation while Gremlin 1 is also related to differentiation in combination with BMPs and its dysregulation results in developmental diseases (2023). Gremlin 1 is overexpressed in various types of human cancers (2426). Recently several studies have demonstrated that CSCs prevent the BMP differentiating effect by secreting Gremlin 1 in glioblastoma, suggesting that Gremlin 1 plays a role in maintenance of CSC properties (27,28). In gynecological malignancies such as ovarian, endometrial and cervical cancers, various techniques to isolate cells with CSC-like properties have been reported. For instance, CD44- or CD133-positive cells are known to be more tumorigenic than negative ones in ovarian cancer (29,30). Similar findings have been suggested for side population (SP) cells in endometrial cancer and ALDH1-positive cells in cervical cancer (3136). Gremlin 1 is also reported to be expressed in cervical cancer compared to normal tissues of the cervix (37). A few studies have investigated the significance of BMPs in cervical cancer (3840), however, there are no studies on the clinico-pathological significance of Gremlin 1 in cervical cancer nor on the function of Gremlin 1 in CSC-like cells of cervical cancer.

Viewing the importance of Gremlin 1 in CSC maintenance and expression of Gremlin 1 in cervical cancer tissue, we hypothesized that Gremlin 1 may be correlated with the prognosis of cervical cancer through maintaining CSC properties. In the present study, we first demonstrated the clinicopathological significance of Gremlin 1 in stage I–II cervical cancer patients and the in vitro promoting effects of Gremlin 1 on CSC properties in cervical cancer cells.

Materials and methods

Patients

The present study was approved by the Institutional Ethics Committee, and written informed consent was obtained from each patient. Patients with a diagnosis of cervical cancer (stage I–II) who underwent primary surgery at the University of Tokyo Hospital from 2005 to 2014 were enrolled. A total of 104 samples were obtained.

RNA extraction and RT-quantitative PCR (RT-qPCR)

Cervical cancer tissues (100–200 mg) were collected and snap frozen with liquid nitrogen and preserved at −80°C. Tissues were homogenized using MagNA Lyser Instrument (Roche Diagnostics, Mannheim, Germany) and total RNA was extracted with a Tissue Total RNA kit (Favorgen Biotech Corp., Ping-Tung, Taiwan) according to the manufacturer's protocols. First-strand cDNA was synthesized (reverse transcription reactions) from 1 µg of total RNA using ReverTra Ace (Toyobo, Osaka, Japan). RT-qPCR was performed with SYBR-Green PCR Master Mix (Roche Diagnostics) according to the manufacturer's instructions. β-actin was used as a housekeeping gene, and the results are represented as fold-change relative to β-actin expression (2−ΔΔCt). The sequences of the primer pairs used were as follows: Gremlin 1, forward, 5′-TCATCAACCGCTTCTGTTACGGC-3′ and reverse, 5′-CAGAAGGAGCAGGACTGAAAGG-3′; Nanog, forward 5′-GCTGAGATGCCTCACACGGAG-3′ and reverse 5′-TCTGTTTCTTGACCGGGACCTTGTC-3′; OCT3/4, forward 5′-TGGAGAAGGAGAAGCTGGAGCAAAA-3′ and reverse 5′-GGCAGATGGTCGTTTGGCTGAATA-3′; SOX2, forward 5′-GGAAATGGGAGGGGTGCAAAAGAGG-3′ and reverse 5′-TTGCGTGAGTGTGGATGGGATTGGTG-3′; β-actin, forward 5′-CTGGAACGGTGAAGGTGACA-3′ and reverse 5′-AAGGGACTTCCTGTAACAACGCA-3′. Denaturation was performed at 95°C for 2 min, followed by 35 cycles at 98°C for 10 sec, at 65°C for 10 sec and at 68°C for 8 sec. Each experiment was performed in triplicate and repeated three times.

Cell culture

The human squamous cell carcinoma cell line CaSki (derived from a metastatic site; small intestine) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in adherent conditions in Dulbecco's modified Eagle's medium (DMEM; Wako Pure Chemical Industries Ltd., Osaka, Japan) supple mented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies, Carlsbad, CA, USA) and sub-cultured by 0.25% trypsin/EDTA (Wako Pure Chemical Industries Ltd.) detachment. For the sphere-forming assays, DMEM/F12 (Invitrogen Life Technologies) supplemented with 20 ng/ml human recombinant epidermal growth factor (EGF; Wako Pure Chemical Industries Ltd.), 10 ng/ml basic fibroblastic growth factor (bFGF; ReproCELL, Inc., Kanagawa, Japan) and 0.3% bovine serum albumin (BSA; Sigma-Aldrich Co., St. Louis, MO, USA) (sphere medium) was used. Each medium contained no antibiotics. All cells were grown and treated in a humidified atmosphere at 37°C and 5% CO2. When 60–70% confluence was reached, the medium was replaced with DMEM containing 0.5% FBS and 1,000 ng/ml human recombinant Gremlin 1 (R&D Systems Inc., Minneapolis, MN, USA) or vehicle for another 24 h and total RNA was extracted.

Flow cytometry

The ALDH enzymatic activity of the cells was measured using the ALDEFLUOR kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's protocol. CaSki cells (1×106 cells) were suspended in ALDEFLUOR assay buffer containing ALDH substrate. The brightly fluorescent ALDH-positive cells were detected using FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). As a negative control, cells were stained under identical conditions after treatment with the specific ALDH inhibitor N,N-diethylaminobenzaldehyde (DEAB). For obtaining consistent results, the following conditions were applied; 2×105 cells were seeded onto a 6-well multi-plate dish in triplicate for 48 h and medium was switched to DMEM containing 3% FBS and Gremlin 1 (1,000 ng/ml) or vehicle for another 24 h and flow cytometry was performed as described above. Each experiment was repeated three times.

Sphere-forming assay

CaSki cells were cultured under the conditions described for flow cytometry, and then the sphere-forming assay was performed. Dissociated single cells (1,000 cells) were seeded onto ultra-low attachment multi-plate (24-well) dish (Corning Inc., Corning, NY, USA) in quadruplicate and cultured in sphere medium for another 7 days. After that, spheroids larger than 100 µm in diameter were counted, and the number of spheroids was compared between the Gremlin 1-exposed cells and the control (vehicle). Each experiment was repeated three times.

Statistical analysis

Known clinicopathological prognostic factors and expression of Gremlin 1 mRNA were analyzed by univariate Cox proportional hazards regression models, and the relative risk (RR) with 95% confidence intervals (CIs) was evaluated. Logistic regression analysis was performed to investigate the relationship between Gremlin 1 mRNA expression level and recurrence. Using this result, receiver operating characteristic (ROC) curves were used to assess the criterion value. Kaplan-Meier curves were used to estimate the probability of OS and PFS, and the log-rank test was used to compare survival between the two groups (high Gremlin 1 mRNA or low Gremlin 1 mRNA levels). A χ2 test was used to evaluate the relationship between expression of Gremlin 1 and each clinicopathological factor. For the in vitro study, the Student's t-test was used to compare the data obtained for the controls. P<0.05 was considered to indicate a statistically significant difference. JMP® (SAS Institute) was used for statistical analysis.

Results

Expression of Gremlin 1 in cervical cancer tissues is significantly correlated with progression-free survival (PFS) and recurrence

Characteristics of the 104 patients whose cancer tissues were collected are shown in Table I. Expression of Gremlin 1 mRNA was not a significant prognostic factor for overall survival (OS) in the univariate analysis (Table II) but was a prognostic factor for PFS in both univariate and multivariate analyses as well as histology (non-squamous carcinoma) (Table III). Expression of Gremlin 1 mRNA was also significantly correlated with recurrence in the logistic regression analysis. Cut-off value for high and low expression was set using ROC curves; high expression of Gremlin 1 mRNA, >0.20; low expression, <0.20 (Fig. 1). Kaplan-Meier curves for OS and PFS in patients with high and low Gremlin 1 mRNA expression are shown in Fig. 2. High expression of Gremlin 1 mRNA was a significant prognostic factor for PFS (log-rank test, P=0.0004).

Table I

Characteristics of the cervical cancer patients.

Table I

Characteristics of the cervical cancer patients.

FeaturesCases (n)Percentage (%)
Mean age (range) in years44.0 (22.9–75.1)
FIGO stage
 Stage I6966
 Stage II3534
Histology
 SCC5956
 Non-SCC4544
Tumor size
 ≤4 cm4644
 >4 cm5856
Pm invasion
 Negative7975
 Positive2525
Stromal invasion
 ≤2/34846
 >2/35654
Margin
 Negative9894
 Positive66
LVSI
 Negative4341
 Positive6159
LN metastasis
 Negative7370
 Positive3130
Recurrence
 Negative8481
 Positive2019

[i] SCC, squamous cell carcinoma; Pm, parametrium; LVSI, lymphovascular space involvement; LN, lymph node.

Table II

Predictive factors for overall survival (OS).

Table II

Predictive factors for overall survival (OS).

FeaturesUnivariate
Multivariate
Relative risk (95% CI)P-valueRelative risk (95% CI)P-value
Stage (II vs. I)2.91 (0.60–16.07)0.17821.44 (0.28–9.15)0.6624
Histology (NS vs. SCC)3.13 (0.66–22.04)0.15084.11 (0.86–29.21)0.0758
Bulky (>4 cm) tumor (positive vs. negative)5.14 (0.86–97.49)0.07391.81 (0.28–35.39)0.5679
Pm invasion (positive vs. negative)0.61 (0.032–3.81)0.6404
Stromal invasion (>2/3 vs. ≤2/3)1.34 (0.27–7.41)0.7114
Margin (positive vs. negative)3.26 (0.16–20.46)0.3458
LVSI (positive vs. negative)1.28e9 (2.23-#)0.0064a1.04e9 (1.31–111)0.0064a
LN metastasis (positive vs. negative)2.02 (0.38–9.65)0.3796
Expression of Gremlin 10.39 (0.017–1.54)0.3354

{ label (or @symbol) needed for fn[@id='tfn2-or-35-01-0391'] } LVSI was statistically significant to predict poor prognosis in the multivariate analysis. Factors showing a tendency of association with poor prognosis (P<0.25) in the univariate analysis were included in the multivariate model. Parametrium invasion and stromal invasion were excluded due to possible multicollinearity with bulky tumor. #, the value was not available in the software.

a P<0.05. NS, non-squamous carcinoma; SCC, squamous cell carcinoma; Pm, parametrium; LVSI, lymphovascular space involvement; LN, lymph node; CI, confidence interval.

Table III

Predictive factors for progression-free survival (PFS).

Table III

Predictive factors for progression-free survival (PFS).

FeaturesUnivariate
Multivariate
Relative risk (95% CI)P-valueRelative risk (95% CI)P-value
Stage (II vs. I)3.30 (1.36–8.43)0.0082a1.84 (0.69–5.27)0.3247
Histology (NS vs. SCC)2.57 (1.05–6.86)0.0379a4.01 (1.55–11.51)0.0077a
Bulky (>4 cm) tumor (positive vs. negative)4.86 (1.63–20.84)0.00302.85 (0.86–13.10)0.1021
Pm invasion (positive vs. negative)2.20 (0.86–5.33)0.0959
Stromal invasion (>2/3 vs. ≤2/3)1.59 (0.65–4.24)0.3108
Margin (positive vs. negative)1.06 (0.05–5.18)0.9491
LVSI (positive vs. negative)6.84 (1.97–43.04)0.0011a3.86 (0.92–26.41)0.0538
LN metastasis (positive vs. negative)2.37 (0.97–5.79)0.0565
Expression of Gremlin 1 mRNA1.43 (1.05–1.78)0.0281a1.41 (1.01–1.78)0.0036a

{ label (or @symbol) needed for fn[@id='tfn4-or-35-01-0391'] } Factors showing a significant association with poor prognosis (P<0.05) in the univariate analysis were included in the multivariate model.

a P<0.05. Histology and expression of Gremlin 1 mRNA were statistically significant to predict poor prognosis in the multivariate analysis.NS, non-squamous carcinoma; SCC, squamous cell carcinoma; Pm, parametrium; LVSI, lymphovascular space involvement; LN, lymph node; CI, confidence interval.

High expression of Gremlin 1 in cervical cancer tissues is significantly correlated with a bulky tumor

The correlations between expression of Gremlin 1 mRNA and each prognosis factor are listed in Table IV. Of note, high expression of Gremlin 1 mRNA was significantly correlated with a bulky (>4 cm) tumor (χ2 test, P=0.0494).

Table IV

Correlation between expression of Gremlin 1 mRNA (high or low) and each risk clinicopathological factor.

Table IV

Correlation between expression of Gremlin 1 mRNA (high or low) and each risk clinicopathological factor.

FeaturesRelative risk (95% CI)P-value
Stage (II vs. I)1.38 (0.81–2.37)0.2325
Histology (NS vs. SCC)1.14 (0.37–1.77)0.5415
Bulky (>4 cm) tumor (positive vs. negative)1.40 (1.001–1.971)0.0494a
Pm invasion (positive vs. negative)1.96 (0.97–3.95)0.0534
Stromal invasion (>2/3 vs. ≤2/3)1.05 (0.74–1.51)0.7600
Margin (positive vs. negative)2.62 (0.50–13.68)0.2345
LVSI (positive vs. negative)1.04 (0.75–1.43)0.8076
LN metastasis (positive vs. negative)1.59 (0.88–2.87)0.0189

{ label (or @symbol) needed for fn[@id='tfn6-or-35-01-0391'] } Correlation between high Gremlin 1 mRNA expression and bulky tumor was significant (χ2 test. P=0.0494).

a P<0.05. NS, non-squamous carcinoma; SCC, squamous cell carcinoma; Pm, parametrium; LVSI, lymphovascular space involvement; LN, lymph node; CI confidence interval.

Exposure of CaSki cells to Gremlin 1 increases Nanog mRNA expression levels and the percentage of ALDH1-positive cells in vitro

We then speculated whether the clinical significance of Gremlin 1 expression is attributed to CSC-like properties. We decided to use a CaSki cell in vitro model as CaSki cells are derived from a recurrent and metastatic site. Cancer stem cells are thought to share normal stem cell features and express undifferentiated-cell markers such as Nanog, Oct3/4 and Sox2. ALDH1-positive cells are reported to be a marker of cancer stem-like cells in various types of solid tumors including cervical cancer. After culturing CaSki cells for 48 h, we exposed cells to Gremlin 1 (1,000 ng/ml) or vehicle control for 24 h and assessed these markers. Exposing CaSki cells to Gremlin 1 significantly increased the expression of Nanog mRNA but not Oct3/4 and Sox2 mRNA (Fig. 3A). Likewise, exposing CaSki cells to Gremlin 1 significantly (~1.41-fold) increased the population of ALDH1-positive cells compared to the control (Fig. 3B). These results were not obtained when the concentration of Gremlin 1 was reduced to 500 ng/ml (data not shown).

Exposing CaSki cells to Gremlin 1 increases sphere-forming ability

We then investigated the sphere-forming ability of Gremlin 1-exposed cells. Significantly more spheroids were formed when 1,000 cells of Gremlin 1-exposed cells were seeded compared to the control (Fig. 4A). A representative counted spheroid (>100 µm in diameter) is shown in Fig. 4B.

Discussion

Here, we investigated the clinical significance of Gremlin 1 in cervical cancer and then investigated its effects on cancer cells in vitro.

The CSC theory states that only a small population of cells within a tumor is tumorigenic and has the ability for self-renewal. Increasing evidence suggests that tumor tissues are not homogeneous but heterogeneous and supports the CSC hypothesis in cervical cancer as well. Recently, Gremlin 1 has been suggested to be secreted by CSCs in glioblastoma and prevents CSCs from differentiating by its antagonistic effect on BMPs.

First, we showed that high expression of Gremlin 1 mRNA in cervical cancer tissues is a significant prognostic factor for PFS and clinical recurrence. High expression of Gremlin 1 mRNA was significantly correlated with a bulky (>4 cm) tumor, which is one of the most malignant features of cervical cancer. However, these findings should not be interpreted to conclude that cases of poor prognosis have a larger population of CSCs. Gremlin 1 was found to be secreted from stromal cancer-associated fibroblast (CAFs) in colon cancer and its expression was localized to invasion fronts (2426). On the other hand, Gremlin 1 was found to be secreted by CSCs in glioblastoma and was thought to prevent CSCs from differentiating by antagonizing the effects ofBMPs (27,28). Although there are no common views about the distribution pattern of Gremlin 1 expression in cervical cancer (37), we may safely say that a microenvironment of high expression of Gremlin 1 is favorable for CSC maintenance or invasion.

In an in vitro study, we revealed the effects of Gremlin 1 on CSC-like properties. The exposure of CaSki cells to Gremlin 1 (1,000 ng/ml) increased: i) Nanog mRNA expression levels (but not Oct3/4 and Sox2 mRNA expression levels), ii) the population of ALDH-positive cells (1.41-fold higher compared with the control) and iii) sphere-forming ability when 1,000 Gremlin 1-exposed cells were seeded.

The effects of Gremlin 1 may have been obtained by preventing CSCs from differentiating or promoting non-CSCs to de-differentiate. We investigated changes in the population of ALDH1-positive CaSki cells in normal culture medium at each day after 48 h of seeding (Fig. 5). After 48 h of seeding, >60% of adherent cells were ALDH1-positive and those percentages were reduced to ~30% upon reaching confluency, which are compatible with previous reports that the ALDH1-positive population in CaSki cells are 20–30% (32). In our experiments of Gremlin 1-exposure, the cells were ~60–70% confluent after 48 h of seeding (when we exposed cells to Gremlin 1) and became 90–100% confluent when assessed after 72 h of seeding. Considering this fact and a previous report that ALDH1-negative cells do not become ALDH-positive (32), the effects of Gremlin 1 that we observed were achieved by preventing CSCs from differentiating to some extent resulting in increased sphere-forming ability.

Gremlin 1 specifically antagonizes BMP2, BMP4 and BMP7 function among the various members of BMPs (19). We performed the same experiments using BMP2, BMP4 and BMP7 (500 ng/ml). Only exposure of CaSki cells to BMP7 significantly reduced the expression of Nanog mRNA expression. However, the population of ALDH1-positive cells was not significantly different even between BMP7-exposed cells and the vehicle control (data not shown). This fact suggests that the BMP/Gremlin 1 axis is complex and we should not only consider high and low expression of BMPs or Gremlin 1 but the combination is important. Gremlin 1 is thought to have various effects on non-BMP signaling (41). We performed the same experiments using LDN-193189 [a small molecule inhibitor of BMP type I receptors ALK2 and ALK3 (4244)] to confirm that our obtained results of Gremlin 1 were via BMP signaling. Exposure of CaSki cells to LDN-193189 increased the expression of Nanog mRNA in a dose-dependent manner and significantly high expression of Nanog mRNA was observed compared to the vehicle control when the concentration of LDN-193189 was 100 nM (data not shown).

In conclusion, we demonstrated the clinical significance of Gremlin 1 in cervical cancer and insights into its effects on CSC-like properties in vitro were shown. In cervical cancer, it is suggested that Gremlin 1 may play a role in clinical recurrence and maintaining CSC-like properties.

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January-2016
Volume 35 Issue 1

Print ISSN: 1021-335X
Online ISSN:1791-2431

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Copy and paste a formatted citation
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Spandidos Publications style
Sato M, Kawana K, Fujimoto A, Yoshida M, Nakamura H, Nishida H, Inoue T, Taguchi A, Takahashi J, Adachi K, Adachi K, et al: Clinical significance of Gremlin 1 in cervical cancer and its effects on cancer stem cell maintenance. Oncol Rep 35: 391-397, 2016
APA
Sato, M., Kawana, K., Fujimoto, A., Yoshida, M., Nakamura, H., Nishida, H. ... Fujii, T. (2016). Clinical significance of Gremlin 1 in cervical cancer and its effects on cancer stem cell maintenance. Oncology Reports, 35, 391-397. https://doi.org/10.3892/or.2015.4367
MLA
Sato, M., Kawana, K., Fujimoto, A., Yoshida, M., Nakamura, H., Nishida, H., Inoue, T., Taguchi, A., Takahashi, J., Adachi, K., Nagasaka, K., Matsumoto, Y., Wada‑Hiraike, O., Oda, K., Osuga, Y., Fujii, T."Clinical significance of Gremlin 1 in cervical cancer and its effects on cancer stem cell maintenance". Oncology Reports 35.1 (2016): 391-397.
Chicago
Sato, M., Kawana, K., Fujimoto, A., Yoshida, M., Nakamura, H., Nishida, H., Inoue, T., Taguchi, A., Takahashi, J., Adachi, K., Nagasaka, K., Matsumoto, Y., Wada‑Hiraike, O., Oda, K., Osuga, Y., Fujii, T."Clinical significance of Gremlin 1 in cervical cancer and its effects on cancer stem cell maintenance". Oncology Reports 35, no. 1 (2016): 391-397. https://doi.org/10.3892/or.2015.4367