Androgens as potential prognostic biomarkers are correlated with E-cadherin/β-catenin pathway in endometrial carcinoma

doi:10.21203/rs.3.rs-4611752/v1

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Androgens as potential prognostic biomarkers are correlated with E-cadherin/β-catenin pathway in endometrial carcinoma

https://doi.org/10.21203/rs.3.rs-4611752/v1

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Background

Androgens are associated with the risk of endometrial carcinoma (EC). However, their roles as prognostic factors are less clear. This study aimed to investigate the prognostic impact of different androgenic hormones and further uncovered the involved mechanism.

Methods

We examined associations between endogenous testosterone levels (total, bioavailable and free testosterone) and the clinical outcomes in patients with EC. The biological function of androgen was examined by in vitro and in vivo experiments.

Results

Compared with healthy controls, patients with EC showed significantly higher levels of testosterone. However, higher levels of bioavailable and free testosterone in EC patients correlated with favourable clinicopathological features. Kaplan-Meier survival analysis indicated that higher bioavailable and free testosterone levels were associated with better overall survival (P = 0.048, P = 0.036, respectively), which was not observed in subgroup analysis of total testosterone (P = 0.097). Furthermore, in vitro and in vivo experiments confirmed that androgen-dihydrotestosterone (DHT) could inhibit the migration and invasion of EC cells. Mechanistically, DHT could recruit β-catenin to E-cadherin to stabilize the adhesion junctions and inhibit β-catenin translocation into the nucleus in EC cells.

Conclusions

Androgens may potentially predict the prognosis of patients with EC.

Androgen

E-cadherin/β-catenin

Endometrial carcinoma

Metastasis

Prognosis

Endometrial carcinoma (EC) is the most common female genital tract cancer(1). It’s well established that an excess-estrogen environment is associated with the risk for EC. Now, accumulating evidence supports the potential of androgens in the carcinogenesis of EC.

The preponderance of epidemiological studies has reported strong associations between elevated levels of testosterone and an increased risk of EC in postmenopausal women(2–6).

However, it’s unclear if androgens influence the prognosis of EC patients. Besides, existing studies exploring circulating androgens and EC focused only on total hormone levels or/and its free fraction in plasma. Testosterone circulates in the bloodstream nonspecifically bound to albumin, specifically bound to sex hormone-binding globulin (SHBG), and only a small part dissolved freely. The SHBG-bound fraction is biologically inactive because of the high binding affinity of SHBG for testosterone. The free and nonspecifically bound plasma hormone levels are commonly identified as the bioavailable fraction and can potentially cross the cellular membranes exerting androgenic actions(7). It’s important to determine which one is the best reliable index of testosterone among three androgenic indicators including bioavailable testosterone, free testosterone and total testosterone,

Further, the mechanism by which androgen (dihydrotestosterone, DHT) exerts related effects in EC remains unknown. The conversion of carcinoma cells from epithelial cells into a mesenchymal-like phenotype (known as epithelial-mesenchymal transition, EMT) is highly important due to its potential role in the promotion of tumor metastasis(8). We determined whether DHT influence the process of EC via EMT-related signaling pathways.

Patient samples and measurements of endogenous sex hormones

A total of 465 patients with EC and 465 controls at Tianjin Medical University General Hospital were included in the study between August 19, 2003, and November 4, 2015. The patients were first diagnosed with histologically confirmed EC. None of them underwent hormonal therapy, radiotherapy, or chemotherapy prior to surgery. Tumor grade, stage [using the surgical staging criteria from the International Federation of Gynecology and Obstetrics, (FIGO)], lymph node status, myometrial invasion, cervical invasion, and ovarian invasion were retrieved by review of hospital notes and clinical databases. By November 2015, the average follow-up time was 37 months, ranging from 1 to 130 months. During the follow-up, 30 deaths occurred (23 due to disease progression and 7 from other reasons). Women who went to Tianjin Medical University General Hospital for a routine examination were used as controls. None of them had a history of cancer, and they were time- and nationality-matched to the patients. The levels of total testosterone were measured using chemiluminescence reagents (Siemens Medical Solutions, PA, USA). SHBG was quantified with the use of sandwich enzyme linked immunosorbent assay (R&D Systems, MN, USA) following the manufacturers’ protocols. The levels of bioavailable and free testosterones were calculated with a confirmed algorithm that used the total testosterone hormone levels, SHBG level, albumin level, and association constants for the binding of testosterone to SHBG and albumin(7, 9). For three androgenic indexes, quartiles 3 and 4 (Q3 and Q4) were labeled as “high level,” Q1 and Q2 were designated as “low level”. All patients provided informed consent before experiments. The Ethical Committee of Tianjin Medical University General Hospital approved this study and performed in a manner consistent with the Declaration of Helsinki (2013).

Cell culture and treatments

EC cells, including HEC-1B (cat. no. 1101HUM-PUMC000105) and KLE (cat. no. CL-0133), are obtained from the headquarters of China Infrastructure of Cell Line Resource, National Sciences & Technology Infrastructure and Procell Life Science& Technology Co.,Ltd respectively. KLE was cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (1:1) (Gibco/Life Technologies, CA, USA) containing 10% fetal bovine serum (FBS). HEC-1B cells were grown in MEM (Gibco/Life Technologies) containing 1mM sodium pyruvate and 1% non-essential amino acids and supplemented with 10% FBS. For drug treatments in the following experiments, 100nM DHT (Solarbio, Beijing, China) was mixed with DMEM/F12 and MEM with 10% FBS for 16 h(10). All cell lines have been authenticated using short tandem repeat profiling within the last three years. All experiments were performed with mycoplasma-free cells.

Cell migration and invasion assay

Migration assays were evaluated using Transwell membranes (Corning, MA, USA) without Matrigel (Corning). A total of 5 × 10⁴ cells of serum-free DMEM/F12 or MEM containing 100nM DHT were added to a chamber with 24-well plates filled with 10% FBS. After stimulation for 16 h, the cells were fixed with 4% paraformaldehyde and stained with crystal violet. The process for the cell invasion assay was the same as that for the migration assay, except that the Transwell membranes were pre-coated with Matrigel.

Western blot analysis

Basic Western blot procedures were conducted as previously described(11) using antibodies against the following proteins: β-catenin, phospho-β-catenin (Ser675), H3, GSK3β, phospho-GSK3β (S9), E-cadherin, N-cadherin, Vimentin and Na-K ATPase (Cell Signaling Technology, MA, USA). The anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from ZSGQ-BIO (Beijing, China).

Immunofluorescence assays

The cells were plated in 24-well plates and treated with 100nM DHT (Solarbio) for 16 h. The assays were performed as previously described(11). Primary antibody used was F-actin (Abcam, Cambridge, UK) and the secondary antibody used was Alex Flour 594 (Invitrogen, CA, USA).

Immunoprecipitation (IP)

The cells after DHT treatment for 16 h were lysed using IP lysis buffer (Beyotime Biotechnology, Shanghai, China). The extracts were incubated with the indicated antibodies (Cell Signaling Technology, MA, USA) overnight at 4℃. After 2 h incubation with protein A/G agarose beads (Santa Cruz Biotechnology, Shanghai, China), immune complexes were washed four times. Normal mouse/rabbit IgG (Cell Signaling Technology) were used as negative controls.

Nude mouse xenograft models and treatment

Five-week-old female BALB/c mice were employed in xenograft studies. A total of 1 × 10⁶ luciferase-labeled HEC-1B cells were injected into the tail vein to induce lung metastasis in the xenograft tumor model. Twenty-four hours after injection, the mice were randomly divided and intraperitoneally injected three times per week for a month with DHT (7 mg/kg) or the same dose of saline for comparison(12). Tumor growth was assessed by bioluminescence imaging once a week. All experimental protocols were carried out according to the Tianjin Medical University guidelines for animal research and approved by the Institutional Animal Care and Use Committee.

Statistical analysis

Data were statistically analyzed with SPSS version 22.0 software, Medcalc and GraphPad Prism 6. In clinical studies, the risk factors for EC were analyzed by conditional logistic regression. Kaplan-Meier survival plots were used to perform survival curves, and the log-rank test was used to compare significance. The chi-squared test was used to identify whether a significant difference existed between high-androgen and low-androgen groups. The two-tailed Student t test or analysis of variance for functional analysis determined statistical significance. The Cox regression model was used to determine independent prognostic factors. Multivariate models included only variables with P < 0.05 in univariate analysis. The error bars in the graphs represented the mean ± standard deviation of at least three independent experiments. P value less than 0.05 was considered statistically significant.

Serum testosterone levels were independent risk factors for EC

The details of the study population were published in Table S1. The significant differences between cases and control subjects in line with known EC risk factors were observed. EC patients had higher concentrations of androgens (total testosterone, free testosterone and bioavailable testosterone), especially total and bioavailable testosterone levels (P<0.001, Table S1). We detected positive associations between EC risk and total/bioavailable testosterone levels via a univariate logistic model. Adjustment for BMI, age and reproduction strengthened the associations of androgens (total testosterone, free testosterone and bioavailable testosterone) with EC risk, suggesting that serum testosterone levels were an independent risk factor for EC (Table 1).

Table 1

Odds ratio for EC risk using logistic regression
	Cases/controls	Unadjusted			Adjusted for^a
	Cases/controls	OR	95%CI	P-value	OR	95%CI	P-value
Total testosterone	465/465	1.016	1.010–1.023	< 0.001	1.014	1.007–1.021	< 0.001
Free testosterone	465/465	1.00	-	1.00	-
Bioavailable testosterone	465/465	1.470	1.341–1.612	< 0.001	1.377	1.253–1.514	< 0.001
^a adjusted for BMI, age and reproduction.
OR: odds ration; CI: confidence interval

High levels of bioavailable and free testosterone were positively associated with favorable prognostic factors

Then testosterone levels were further classified into two groups (high level and low level). The correlations between the levels of different testosterone indexes and clinicopathological parameters were summarized in Table 2. A statistically significant trend of favorable prognosis with higher levels of free and bioavailable testosterone level was found among patients with EC. The levels of bioavailable and free testosterone were inversely associated with the FIGO stage (stages I and II vs stages III and IV, P = 0.014 and 0.012 respectively) and lymph node status (P < 0.001). A statistically significant correlation was observed between testosterone levels (bioavailable and free androgen) and myometrial invasion (85.8% of patients in the high testosterone group had no more than one half myometrial invasion, P = 0.001), which was coupled with bioavailable/free testosterone and cervical invasion (nearly 94.0% of patients in the high testosterone group were negative for a cervical invasion at the time of diagnosis, P = 0.018, 0.023 respectively).

Table 2

Association of bioavailable testosterone, free testosterone and total testosterone with clinicopathologic parameters in EC
Clinical characteristics						Free testosterone
Clinical characteristics	Low	High	χ²	P	Low	High	χ²	P	Low	High	χ²	P
Age (y)
≤ 50	41(50.0%)	41(50.0%)	0.104	0.747	42(51.2%)	40(48.8%)	0.070	0.791	45(54.9%)	37(45.1%)	0.678	0.410
>50	199(52.0%)	184(48.0%)	0.104	0.747	190(49.6%)	193(50.4%)	0.070	0.791	191(49.9%)	192(50.1%)	0.678	0.410
BMI^a
<24	82(68.3%)	38(31.7%)	18.334	< 0.001	79(65.8%)	41(34.2%)	16.676	< 0.001	68(56.7%)	52(43.3%)	2.347	0.126
≥ 24	157(45.6%)	187(54.4%)	18.334	< 0.001	152(44.2%)	192(55.8%)	16.676	< 0.001	167(48.5%)	177(51.5%)	2.347	0.126
Reproduction
Never	25(56.8%)	19(43.2%)	0.527	0.468	25(56.8%)	19(43.2%)	0.932	0.334	26(59.1%)	18(40.9%)	1.352	0.245
Ever	215(51.1%)	206(48.9%)	0.527	0.468	207(49.2%)	214(50.8%)	0.932	0.334	210(49.9%)	211(50.1%)	1.352	0.245
Histological type
Endometrioid	203(50.4%)	200(49.6%)	1.863	0.172	195(48.4%)	208(51.6%)	2.740	0.098	196(48.6%)	207(51.4%)	5.422	0.020
Non-endometrioid	37(59.7)	25(40.3%)	1.863	0.172	37(59.7)	25(40.3%)	2.740	0.098	9(56.3%)	7(43.8%)	5.422	0.020
FIGO Grade
Low (1 + 2)	190(49.7%)	192(50.3%)	3.012	0.083	184(48.2%)	198(51.8%)	2.547	0.110	188(49.2%)	194(50.8%)	2.025	0.155
High (3)	50(60.2%)	33(39.8%)	3.012	0.083	48(57.8%)	35(42.2%)	2.547	0.110	48(42.2%)	35(57.8%)	2.025	0.155
FIGO Stage
Ⅰ+Ⅱ	195(49.2%)	201(50.8%)	6.005	0.014	188(47.5%)	208(52.5%)	6.240	0.012	194(49.0%)	202(51.0%)	3.318	0.069
Ⅲ+Ⅳ	45(65.2%)	24(34.8%)	6.005	0.014	44(63.8%)	25(36.2%)	6.240	0.012	42(60.9%)	27(39.1%)	3.318	0.069
Lymph node status
Negative	208(48.9%)	217(51.1%)	14.121	< 0.001	201(47.3%)	224(52.7%)	13.343	< 0.001	210(49.4%)	216(50.6%)	3.554	0.059
Positive	32(80.0%)	8(20.0%)	14.121	< 0.001	31(77.5%)	9(22.5%)	13.343	< 0.001	26(65.0%)	14(35.0%)	3.554	0.059
Myometrial invasion
No more than half	177(47.8%)	193(52.2%)	10.335	0.001	170(45.9%)	200(54.1%)	11.283	< 0.001	174(47.0%)	196(53.0%)	10.06	0.002
More than half	63(66.3%)	32(33.7%)	10.335	0.001	62(65.3%)	33(34.7%)	11.283	< 0.001	62(65.3%)	33(34.7%)	10.06	0.002
Cervical invasion
No	211(49.9%)	212(50.1%)	5.620	0.018	204(48.2%)	219(51.8%)	5.196	0.023	212(50.1%)	211(49.9%)	0.754	0.385
Yes	29(69.0%)	13(31.0%)	5.620	0.018	28(66.7%)	14(33.3%)	5.196	0.023	24(57.1%)	18(42.9%)	0.754	0.385
Ovarian invasion
No	225(51.1%)	215(48.9%)	0.744	0.388	217(49.3%)	223(50.7%)	1.080	0.299	224(50.9%)	216(49.1%)	0.080	0.777
Yes	15(60.0%)	10(40.0%)	0.744	0.388	15(60.0%)	10(40.0%)	1.080	0.299	12(48.0%)	13(52.0%)	0.080	0.777
^a BMI data were available for only 464 cases.

This study also examined the similar association between total serum testosterone and clinicopathological characteristics, as shown in Table 2. In contrast, lower total testosterone levels were observed only in patients with myometrial invasion (P = 0.002).

Bioavailable and free testosterone influenced patient outcomes

Generally, patients with high levels of androgens showed a longer overall survival (OS) than those with low levels of androgens (Fig. 1). A significant reduction in OS was observed in the low level of bioavailable testosterone (112.790 ± 3.937 vs. 124.144 ± 3.059, P = 0.048) and free testosterone (112.444 ± 3.997 vs. 124.252 ± 3.033, P = 0.036) (Fig. 1a and 1b). Patients with high total testosterone also had a longer mean OS (123.265 ± 3.256 vs. 112.883 ± 4.029). However, the difference between the two groups was not significant (P = 0.097) (Fig. 1c). The univariate analysis showed that high FIGO grade (G3), advanced stages (Ⅲ + Ⅳ), positive lymph node status, cervical invasion, ovarian invasion and low level of bioavailable testosterone were significantly associated with progressive disease (Table 3). Furthermore, the Cox regression model found that only tumor grade (P = 0.051) and stage (P < 0.001) were independent prognostic indicators (Table 3).

Table 3

Univariable and multivariate analyses of overall survival in EC
Variables	Univariate	Multivariate analysis^a		Multivariate analysis^b
Variables	P-value	P-value	HR (95% CI)	P-value	HR (95% CI)
Age ≤50 vs. >50	0.637	Removed	——	Removed		——
BMI < 24 vs. ≥24	0.295	Removed	——	Removed		——
Reproduction Never vs. ever	0.981	Removed	——	Removed		——
FIGO Grade G1་G2 vs. G3	0.002	0.051	2.710 (0.997–7.367)	0.051		2.710 (0.997–7.367)
FIGO Stage Ⅰ+Ⅱ vs.Ⅲ+Ⅳ	< 0.001	< 0.001	5.864 (2.367–14.531)	< 0.001		5.864 (2.367–14.531)
Lymph node status Negative vs. positive	< 0.001	0.565	——	0.565		——
Cervical invasion No vs. yes	0.009	0.104	——	0.104		——
Ovarian invasion No vs. yes	0.001	0.545	——	0.545		——
Bioavailable testosterone Low vs. high	0.048	0.312	——	Removed		——
Free testosterone Low vs. high	0.036	Removed	——	0.272		——
Serum total testosterone Low vs. high	0.097	Removed	——	Removed		——
HR = hazard ration; CI = confidence interval
^aThe model was adjusted for FIGO Grade, FIGO Stage, lymph node status, cervical invasion, ovarian invasion and bioavailable testosterone.
^bThe model was adjusted for FIGO Grade, FIGO Stage, lymph node status, cervical invasion, ovarian invasion and free testosterone.

DHT inhibited the motility of EC cells and suppressed EC metastasis in vivo

EC cells in the basal medium were treated with 100nM DHT for 16 h to further confirm whether androgen affected EC progression. The results showed that DHT significantly repressed the migration (52.00 ± 3.362 vs. 18.00 ± 4.506) and invasion (43.20 ± 2.939 vs. 19.80 ± 2.672) of KLE cells (Fig. 2a) (P = 0.0003 and 0.0004, respectively). Also, migration (85.63 ± 4.660 vs 58.13 ± 2.524) and invasion (65.40 ± 5.845 vs. 39.60 ± 3.219) dramatically reduced in HEC-1B cells (Fig. 2b) (P = 0.0001 and 0.0048, respectively). Besides, lung metastasis was induced in xenograft tumor models using HEC-1B cells. In these models, DHT treatment could reduce the generation of metastatic tumor in the lungs, through insignificantly (Fig. 3). These data demonstrated that DHT inhibited the motility of EC cells.

Given the role of EMT in tumor metastasis, we further assessed the relationship between DHT and EMT in EC cells. DHT treatment led to higher levels of E-cadherin and lower levels of mesenchymal markers (Vimentin and N-cadherin). However, DHT had no significant impact on the E-cadherin expression in HEC-1B cells (Fig. 4a). The impact of DHT on lamellipodia was also examined. After 100nM DHT treatment, an obvious reduction in lamellipodia formation was observed (Fig. 4b). These data demonstrated that DHT inhibited the motility of EC cells.

DHT inhibited the oncogenic role of β-catenin via strengthening the E-cadherin/β-catenin complex and upregulation of GSK3β

To further investigated the mechanism of DHT-regulated EMT in EC, the interaction between E-cadherin and β-catenin was examined. The changes in the β-catenin/E-cadherin complex were examined by IP assay using anti-β-catenin and anti-E-cadherin antibodies. The levels of E-cadherin-associated β-catenin and E-cadherin pulled down by the anti-β-catenin antibody increased after 16 h 100nM DHT treatment (Fig. 5a and 5b). Furthermore, we tested whether the strengthening of the E-cadherin/β-catenin complex could contribute to the redistribution of β-catenin. Membrane and nuclear proteins were isolated, and the Western blot analysis showed that the level of β-catenin on the membrane increased and nuclear β-catenin expression decreased (Fig. 5c and 5d). Collectively, these findings confirmed that DHT could induce β-catenin translocation to the cell membrane and stabilize the interaction of β-catenin and E-cadherin.

On the basis of the downregulation of nuclear β-catenin, we further investigated whether DHT involved WNT/β-catenin inactivation. As shown in Fig. 5e, a decrease in the expression levels of β-catenin and p-β-catenin (Ser675) in total proteins was observed after DHT treatment. Moreover, DHT could increase the GSK3β protein expression. These results indicated that DHT was a negative regulator of β-catenin by influencing GSK3β.

Taken together, these results demonstrated that DHT could induce β-catenin binding to E-cadherin and its translocation from nuclei to membrane, thereby resulting in the inactivation of WNT signaling.

The importance of androgens in EC has been recognized since the last century. Increased levels of androgens were associated with an increased risk of EC. We have previously shown that androgens were risk factors of EC(2). The data presented here highlight the importance of uncovering the role that androgens exert on EC prognosis. To our knowledge, this is the first study to show the prognostic value of androgens in patients with EC.

In this study, we re-evaluated the association between androgens and EC risk. Given that the different forms of circulating testosterone, bioavailable testosterone and free testosterone were added in the analysis except for total testosterone. Our observation of increased risk for EC with increasing androgens is consistent with the previous epidemiological evidence(2–6). However, these results are not enough to reveal the effect of androgens in EC due to the lack of the prognosis data. Therefore, we evaluated the association between different levels of androgens and tumor parameters. Interestingly, compared with low levels of androgens, elevated levels of androgens denote a survival advantage. These results suggest us that androgen-regulated signaling may have both oncogenic and tumor suppressive roles. Oncogenic role of androgens maybe more involved in EC initiation. Later stages of invasion and metastasis in EC maybe partly involved in inactivation of cancer suppressive androgen signaling.

Circulating testosterone in plasma is mostly bound to SHBG and the SHBG-bound fraction is biologically inactive because of the high binding affinity of SHBG for testosterone(13, 14). Previous studies on circulating androgens and EC are mostly confined to total serum testosterone. Although free, bioavailable and total testosterone all represent the indexes of testosterone, our clinical data indicate that free testosterone generally reflects the clinic situation most accurately among three androgenic indexes when evaluating prognosis. And it’s the best indicative of the levels of bioactive androgen in EC. Given that the marginal significance of bioavailable testosterone in the analysis of OS, we will continue to follow up to fully demonstrate the role of it in EC.

Although androgens confer better survival on EC patients, they aren’t independent prognostic indicators. The role of androgens in endometrium is complex and likely depends on the interaction with other hormones like estrogens. Some studies suggest that elevated levels of androgens influence endometrium carcinogenesis through their conversion to estrogens(3, 5). Besides, there also exists the synergy between insulin and androgens in EC(2). Unlike in vitro studies, androgen doesn’t significantly inhibit the tumor metastasis in animal models. We cannot rule out the possibility that other hormones in body such as estrogens and insulin interfere with the effect of androgens.

EMT is involved in diverse processes including metastasis, chemoresistance, and tumor stemness(15). The activation of β-catenin or the release of β-catenin from the E-cadherin/β-catenin complex can result in EMT and promote the metastasis of various cancers(16, 17). In fact, studies have demonstrated the involvement of EMT in androgen-regulated signaling in bladder cancer(18). In an attempt to explore whether the inhibitory activity of androgen could be associated with the role of EMT-related pathways in EC, the present study found that androgen could significantly inhibit the motility of EC cells combined with the overexpression of E-cadherin and loss of mesenchymal markers. During EMT, the remodeling of actin and the formation of invasive structures are induced. Lamellipodia, one of the invasive structures, could contribute to the generation of the driving force for cell motility(19). Our results showed that DHT treatment inhibited lamellipodia formation.

β-Catenin could protect a PEST sequence motif on E-cadherin from degradation by a ubiquitin ligase that labeled E-cadherin(20). E-cadherin sequesters β-catenin from the T-cell factor family of transcription factors to the cytoplasmic side of the membrane, hence suppressing the Wnt pathway(21). The data also demonstrated an increased interaction between E-cadherin and β-catenin in response to androgen. β-catenin was not only translocated to the cellular membrane enhancing the structural stability of the cell-cell adhesion junctions, but also pumped out of the nucleus, resulting in the inhibition of the Wnt pathway. In summary, this study provided an insight into the role of androgen in EC. Although androgens were risk factors of EC, elevated levels of androgens were favorable prognostic factors in EC. The most reliable index of androgen was free testosterone rather than total or bioavailable testosterone. Mechanically, androgen inhibited cancer metastasis by recruiting β-catenin to E-cadherin to stabilize the adhesion junctions and inhibiting β-catenin translocation into the nucleus (Fig. 5f).

Our study uncovered that androgens are not only positively associated with EC risk, but also correlate positively favorable prognostic factors by regulating the E-cadherin/β-catenin pathway. In addition, the free levels of testosterone may be the best indicator of the bioactive testosterone concentrations.

DHT, dihydrotestosterone; EC, endometrial carcinoma; EMT, epithelial-mesenchymal transition; OS, overall survival; SHBG, sex hormone-binding globulin;

Acknowledgements

We are thankful to the Key Laboratory of Female Reproductive Health and Eugenics of Tianjin Medical University General Hospital for the support of writing this article.

Ethical approval and consent to participate

The Ethical Committee of Tianjin Medical University General Hospital approved this study (NO. IRB2015-YX-009). All methods were performed in accordance with the relevant guidelines and regulations. This study was performed in accordance with the Declaration of Helsinki.

Consent for publication

We have obtained consent to publish from the participant to report individual patient data.

Availability of data and materials

The data related to the conclusions of this article were all included within the article and its attached files.

Authors’ contributions

Wenyan Tian and Yingmei Wang contributed to the study design and supervision. Xirong Wu performed the research. Kaiwen Zhang wrote the manuscript. Tianqi Wang and Jingying Zhang provided clinical input. Ye Yan assisted with the clinical data analysis and interpretation. Shuangshuang Zhao and Xiaoqing Yang contributed unpublished reagents/analytical tools. All authors read and approved the final manuscript.

Competing interests

All authors declare no conflict of interest.

Funding

This study was funded by the National Natural Science Foundation of China (no. 82172626).

Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer Statistics, 2021. CA: a cancer journal for clinicians. Jan 2021;71(1):7-33. doi:10.3322/caac.21654
Teng F, Ma X, Yu X, et al. High serum Androgen and Insulin concentrations increase the tendency of Endometrial Carcinoma. Journal of Cancer. 2020;11(19):5656-5664. doi:10.7150/jca.46391
Michels KA, Brinton LA, Wentzensen N, et al. Postmenopausal Androgen Metabolism and Endometrial Cancer Risk in the Women's Health Initiative Observational Study. JNCI cancer spectrum. Sep 2019;3(3):pkz029. doi:10.1093/jncics/pkz029
Audet-Walsh E, Lépine J, Grégoire J, et al. Profiling of endogenous estrogens, their precursors, and metabolites in endometrial cancer patients: association with risk and relationship to clinical characteristics. The Journal of clinical endocrinology and metabolism. Feb 2011;96(2):E330-9. doi:10.1210/jc.2010-2050
Lukanova A, Lundin E, Micheli A, et al. Circulating levels of sex steroid hormones and risk of endometrial cancer in postmenopausal women. International journal of cancer. Jan 20 2004;108(3):425-32. doi:10.1002/ijc.11529
Allen NE, Key TJ, Dossus L, et al. Endogenous sex hormones and endometrial cancer risk in women in the European Prospective Investigation into Cancer and Nutrition (EPIC). Endocrine-related cancer. Jun 2008;15(2):485-97. doi:10.1677/erc-07-0064
Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. The Journal of clinical endocrinology and metabolism. Oct 1999;84(10):3666-72. doi:10.1210/jcem.84.10.6079
Chaffer CL, San Juan BP, Lim E, Weinberg RA. EMT, cell plasticity and metastasis. Cancer metastasis reviews. Dec 2016;35(4):645-654. doi:10.1007/s10555-016-9648-7
Rinaldi S, Geay A, Dechaud H, et al. Validity of free testosterone and free estradiol determinations in serum samples from postmenopausal women by theoretical calculations. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. Oct 2002;11(10 Pt 1):1065-71.
Chia KM, Liu J, Francis GD, Naderi A. A feedback loop between androgen receptor and ERK signaling in estrogen receptor-negative breast cancer. Neoplasia (New York, NY). Feb 2011;13(2):154-66. doi:10.1593/neo.101324
Wang Y, Hu L, Ji P, et al. MIIP remodels Rac1-mediated cytoskeleton structure in suppression of endometrial cancer metastasis. Journal of hematology & oncology. Oct 19 2016;9(1):112. doi:10.1186/s13045-016-0342-6
Zhu A, Li Y, Song W, et al. Antiproliferative Effect of Androgen Receptor Inhibition in Mesenchymal Stem-Like Triple-Negative Breast Cancer. Cell Physiol Biochem. 2016;38(3):1003-14. doi:10.1159/000443052
Manni A, Pardridge WM, Cefalu W, et al. Bioavailability of albumin-bound testosterone. The Journal of clinical endocrinology and metabolism. Oct 1985;61(4):705-10. doi:10.1210/jcem-61-4-705
Vermeulen A, Stoïca T, Verdonck L. The apparent free testosterone concentration, an index of androgenicity. The Journal of clinical endocrinology and metabolism. Nov 1971;33(5):759-67. doi:10.1210/jcem-33-5-759
Loh CY, Chai JY, Tang TF, et al. The E-Cadherin and N-Cadherin Switch in Epithelial-to-Mesenchymal Transition: Signaling, Therapeutic Implications, and Challenges. Cells. Sep 20 2019;8(10)doi:10.3390/cells8101118
Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. Nov 3 2006;127(3):469-80. doi:10.1016/j.cell.2006.10.018
Gumbiner BM. Regulation of cadherin-mediated adhesion in morphogenesis. Nature reviews Molecular cell biology. Aug 2005;6(8):622-34. doi:10.1038/nrm1699
Gil D, Zarzycka M, Dulińska-Litewka J, Ciołczyk-Wierzbicka D, Lekka M, Laidler P. Dihydrotestosterone increases the risk of bladder cancer in men. Human cell. Jul 2019;32(3):379-389. doi:10.1007/s13577-019-00255-3
Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion. Cancer metastasis reviews. Jun 2009;28(1-2):15-33. doi:10.1007/s10555-008-9169-0
Hinck L, Näthke IS, Papkoff J, Nelson WJ. Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly. The Journal of cell biology. Jun 1994;125(6):1327-40. doi:10.1083/jcb.125.6.1327
Park SY, Shin JH, Kee SH. E-cadherin expression increases cell proliferation by regulating energy metabolism through nuclear factor-κB in AGS cells. Cancer science. Sep 2017;108(9):1769-1777. doi:10.1111/cas.13321

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Androgens as potential prognostic biomarkers are correlated with E-cadherin/β-catenin pathway in endometrial carcinoma

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Material and Methods

Patient samples and measurements of endogenous sex hormones

Cell culture and treatments

Cell migration and invasion assay

Western blot analysis

Immunofluorescence assays

Immunoprecipitation (IP)

Nude mouse xenograft models and treatment

Statistical analysis

Results

Serum testosterone levels were independent risk factors for EC

High levels of bioavailable and free testosterone were positively associated with favorable prognostic factors

Bioavailable and free testosterone influenced patient outcomes

DHT inhibited the motility of EC cells and suppressed EC metastasis in vivo

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

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