The Role Of Tumor-associated Macrophages In Regulating Estrogen Sensitivity Of Endometrial Carcinoma And Its Mechanism

The role of tumor-associated macrophages in regulating estrogen sensitivity of endometrial carcinoma and its mechanismEndometrial carcinoma (EC) is one of the most commonly diagnosed gynecologic malignancies. Approximately 70-80% of EC cases are type Ⅰ endometrial carcinoma (adenocarcinoma), of which unopposed estrogen exposure is widely regarded as the key mechanism of oncogenesis. Our previous study found that there was not significant difference in serum estradiol levels in type Ⅰ EC and its precancerous lesion patients compared with healthy women of the same age. This finding suggests that there might exist some other mechanisms that participate in the initiation and progression of type I EC.Piles of studies have shown that the key role of inflammation in oncogenesis and development of many malignant tumors. This might also be true in endometrial carcinoma. Epidemiologic studies demonstrated the close relationships between type I EC and insulin resistance (IR), while inflammation is one of the most important clinical manifestations of the later. Clinical studies also demonstrated that, as a specific manifestation of chronic inflammation, macrophages infiltration was positively related with poor prognosis in EC. These findings remind us that macrophage infiltration might play a key role in oncogenesis and progression of type ⅠEC. But the true role of macrophage infiltration and its possible mechanism remains to be investigated.Human monocytes/macrophages can be induced to two subtypes:type 1(M 1) and type 2 (M2). M2 macrophage is the main type of macrophage which infiltrates in malignant tumor lesions, which also known as tumor-associated macrophages (TAMs). Cancer lesion TAMs infiltration is related to poor prognosis of many cancers. Studies have shown that TAMs could moderate the hormone receptor expression in cancer cells. In prostate cancer, TAMs infiltration could up-regulate androgen receptor (AR) expression, and promote the carcinogenic function of AR. In breast cancer, the chronic inflammation state resulting from obesity could activate estrogen receptor in cancer cells.According to all these findings, we make the hypothesis that TAMs infiltration in endometrial lesions might up-regulate estrogen sensitivity of endometrial cancer cells which might play a key role in oncogenesis and development of endometrial cancer.To demonstrate our hypothesis we did both clinical and in vitro studies. In clinical study, we analyzed the relevancy of macrophages infiltration with EC and its precancerous disorders. In in vitro stuy, we investigated the effect of TAMs on regulating estrogen receptor expression and estrogen sensitivity in endometrial cancer cells. Our study included four parts: ①The analysis of serum estradiol levels in type Ⅰ EC and endometrial hyperplasia women; ②The correlation analysis between macrophages infiltration and type Ⅰ EC, endometrial hyperplasia diseases; ③The effect of TAMs on regulating estrogen receptor expression and estrogen sensitivity in endometrial cancer cells; ④ The mechanism of TAMs in regulating ERa expression in endometrial cancer cells.Part Ⅰ:The analysis of serum estradiol levels in type Ⅰ EC and endometrial hyperplasia patientsObjective:To analyze the correlation between the serum estradiol levels and the risk of type Ⅰ EC.Methods:1. Serum estradiol evalution of endometrial cancer and its precancerous diseases401 cases were enrolled from Obstetrics and Gynecology Hospital of Fudan University from September 2011 to December 2013. All the patients received dilation and curettage. The diagnosis were confirmed by pathological diagnosis of endometrial sample, The patients included 69 cases of disordered proliferative endometrium (DPE),168 cases of simple hyperplasia (SH),67 cases of complex hyperplasia (CH),33 cases of endometrial atypical hyperplasia (EAH) and 25 cases of type Ⅰ endometrial carcinoma(EC).39 normal cases were used as control (CTL). The general information of the patients were collected. The serum estradiol level was evaluated by Elisa in the same lab of the hospital. Statistic analysis was done with one-way ANOVA by Graph pad prism 5.0, and p-value=0.05 was chosen as significance test standard.2. Serum estradiol evaluation in endometrial cancer patients and their age paired controlSerum of 68 cases were collected from Obstetrics and Gynecology Hospital of Fudan University from September 2011 to December 2013, including 34 cases of normal control (CTL) and 34 cases of type Ⅰ EC, and the diagnosis were all confirmed by curettage pathology. The ages of normal control and EC cases were pair matched. Serum estradiol levels were tested using ELISA. Statistics analysis was done with paired t test by Graph pad prism 5.0, andp-value=0.05 was chosen as significance test standard.Results:1. Serum estradiol levels distribution in each group:CTL:95.00±68.81ng/L; DPE:151.8±163.2ng/L;SH:156.4±178.8ng/L; CH:138.4±330.0ng/L; EAH:125.5±156.7 ng/L; type Ⅰ EC:36.52±45.35 ng/L, P>0.05. No statistical significant differences were found in serum estradiol level within different groups. The cases were split at age-boundaries into two groups:25～40 year-old group and 40～55 year-old group. Serum estradiol levels in 25～40 year-old cases were CTL:107.1±70.73ng/L; DPE:140.3±165.2ng/L; SH:122.1±129.6ng/L; CH:76.59±59.86ng/L; EAH:120.1±179.2 ng/L, P>0.05. While in 40～55 year-old group, serum estradiol levels were CTL:102.2±63.36ng/L; DPE:161.1±154.5ng/L; SH: 164.0±20.6ng/L; CH:148.1±152.6ng/L; EAH:148.3±122.5 ng/L; type Ⅰ EC:76.86±68.13 ng/L, P>0.05. The results found no statistical significant differences in serum estradiol level within each group.2. Compared serum estradiol levels in age-paired CTL and type Ⅰ EC:P=0.8338. No statistical significant differences in serum estradiol level were found in type Ⅰ EC and age matched control group.Conclusions:No statistical significant differences in serum estradiol level were found in type Ⅰ EC, hyperplasic endometrial disease and normal control after correction of age. The result suggested that beside the widely accepted unopposed estrogen exposure theory, there might exist some other mechanisms which play role in the oncogenesis and development of type Ⅰ EC.Part Ⅱ:The correlation analysis between macrophages infiltration and type Ⅰ EC、 endometrial hyperplasia diseasesObjective:To evaluate the correlation between macrophage infiltration and endometrial hyperplasic diseases and type I ECMethods:20 patients in each of the following group were collected:proliferative endometrium, secretory endometrium, simple hyperplasia and complex hyperplasia.30 atypical hyperplasia and 30 type I endometrial cancer patients were also collected. The endometrial tissues of all the cases above were analyzed., CD68(+) macrophage cell infiltration were investigated using immunohistochemical methods. CD68(+) cell number in each group were counted as well as cell morphology and infiltration site were evalutaed. Statistics analysis was done with one-way ANOVA by Graph pad prism 5.0, and p-value=0.05 was chosen as significance test standard.Results:1. CD68 expressed in macrophage cytoplasm and cell membrane.2. CD68(+) cell number distribution were (per 400*field):proliferative endometrium:6.641±5.463; secretory endometrium:13.82±10.36; simple hyperplasia:20.45±10.17; complex hyperplasia: 35.70±14.73;atypical hyperplasia:41.98±15.74;type Ⅰ endometrial cancer tissue 57.80±25.72, P<0.001 versus type Ⅰ endometrial cancer. CD68 (+) cells number and the progress of endometrial hyperplasia disorders was positively correlated.3. Macrophage infiltration sites progressed from perivascular, interstitial to glandular epithelium with the development of endometrial hyperplasic disease to cancer.4. Macrophage cell morphology tends to be mature with disease progress.Conclusions:CD68(+) macrophage infiltration is positively correlated with progression of endometrial hyperplasic diseases to cancer.Part Ⅲ:TAMs regulate estrogen sensitivity of type Ⅰ EC though ERaObjective:Study the role of TAMs in promoting type Ⅰ EC proliferation and estrogen sensitivity, and analyze further the possible mechanisms.Methods:Two estrogen receptor (ER) (+) type Ⅰ endometrial carcinoma cell lines were used:Ishikawa and Hec-1-A. M2 macrophages which also known as TAMs was induced from monocyte line THP-1. EC cells were co-cultured with THP-1(M2) or cultured in the conditional medium (CM) of THP-1 (M2). The proliferation and estrogen sensitivity of EC cells before and after co-culturing with THP-1(M2) or CM were tested using CCK-8 technology. Physiological estrogen doses (10-9M) was used for estrogen sensitivity test. EC cell proliferation was also evaluated before or after over-expression of ERa or using ER inhibitor ICI. Statistics analysis was done with one-way ANOVA by Graph pad prism 5.0, and p-value=0.05 was chosen as significance test standard. The changes of ERa expression under CM were detected by real-time PCR and Western blot. And cyclinD1 expression was detected by Western blot to confirm the EC cell proliferation changes.Results:1. Human THP-1 (M2) was successfully induced. This was confirmed by morphological chang with pseudopodia, and CD68, CD 163, CD204 and CD206 change in the cells.2. The proliferative abilities and estrogen sensitivities of Ishikawa and HEC-1-A were up-regulated when co-cultured with THP-1 (M2) cells, and the phenomenon could be inhibited by ICI.3. CM of THP-1 (M2) cells could time-dependently promote Ishikawa and HEC-1-A proliferation, and up-regulat the proliferative stimulation ability of estrogen in physiological dose (10-9M), whose function could also be inhibited by ICI.4. CM could time-dependently up-regulate ERa and cyclinD1 expression, but not ER0.5. The proliferation of EC cells after co-cultured with CM was comparable with the proliferation after over-expression with ERa in EC cells.Conclusions:TAMs could promote type Ⅰ EC proliferation and estrogen sensitivity, which was achieved by up-regulating ERa expression in EC cells.Part IV:TAMs regulate ERa expression of type Ⅰ EC cells though IL-17Objective:We screened inflammatory cytokines in TAMs which might play roles in promoting endometrial cells proliferation, and studied their role in pro-proliferation and estrogen sensitivity in EC cells.Methods:After co-cultured with HEC-1-A in Transwell plates, RNA of THP-1(M2) cells or HEC-1-A cells were extracted to screen cytokines using real-time PCR. The selected cytokines after screening were studied to test their effect on EC proliferation and estrogen sensitivity using CCK-8. The effect of the cytokines on ERa expression and p-AKT pathway activation was also evalutated by real-time PCR and Western blot. Statistics analysis was done with one-way ANOVA by Graph pad prism 5.0, and p-value=0.05 was chosen as significance test standard.Results:1. IL-10 and IL-17 were selected after screening.2. Both IL-10 and IL-17 could up-regulate the pro-proliferative ability of estrogen in HEC-1-A, while IL-17 showed stronger function than IL-10.3. Both IL-10 and IL-17 could up-regulate ERa expression in HEC-1-A, and IL-17 showed stronger function, too. So IL-17 was selected to perform the further studies.4. After treated with IL-17 and E2 together, p-AKT signaling pathway was activated in HEC-1-A, which was similar with the finding in EC cells after ERa over-expression.Conclusions:TAMs could up-regulate ERa expression in EC cells though IL-17, which further promote estrogen sensitivity of EC cells, and thus strengthen the pro-proliferative function of estrogen.These results suggest that:1.There was no significant correlation between serum estradiol levels and type I EC progress, unopposed estrogen stimulation might not be the only mechanism in oncogenesis of EC.2. Macrophages infiltration was positively correlated with endometrial hyperplasic diseases and type Ⅰ EC, which might play a key role in the initiation and progression of type I EC.3. TAMs improved the pro-proliferation function of estrogen in EC cells through up-regulating ERa.4. By secreting inflammatory cytokines, like IL-17, TAMs up-regulate ERa expression in EC cells, and further up-regulate their estrogen sensitivity.Our research showed that:Long-term unopposed estrogen stimulation might not be the only mechanism in oncogenesis of type Ⅰ EC. TAMs infiltration which up-regulates estrogen sensitivity of endometrial cancer cells might play a role in oncogenesis and development of type Ⅰ EC. This is achieved by TAMs-IL-17-ERa-pAKT pathway. Targeted anti-inflammation treatment may provide new options for preventing type Ⅰ endometrial carcinoma and precancerous lesions.