Effects And Mechanisms Of Regulation Of Progestin Action By PI3K/Akt Pathway In Endometrial Carcinoma

Endometrial cancer is the most common cancer in the female reproductive system and up to 75-85% of cases are adenocarcinoma (typeⅠendometrial cancer). Progestins were applied in the conservative endocrine treatment to young patients of endometrial adenocarcinoma in order to preserve their fertility. To date, more than 30% of patients with early stage endometrial cancers did not respond to progestin due to de novo or acquired progestin resistance during progestin treatment. Hysterectomy is the only choice for this group of patients to reduce the risk of possible cancer invasion. Currently, continuous efforts are being made to find a way to reverse progestin resistance and to improve the effect of fertility-sparing treatment for endometrial cancer. Various studies reported associations between endometrial cancer risk and individual components of the metabolic syndrome (MetS), which indicates a direct association between various MetS components with the risk of endometrial cancer and progestin resistance.The classical pathway of progesterone is to inhibit endometrial cancer cell proliferation and to reverse their malignant biological behavior by binding to its nuclear progesterone receptor (PR). Upon binding hormone, the receptor changes conformation, allowing dissociation of heat shock proteins and dimerization of two activated receptors. The activated dimer complex preferentially binds to progesterone response elements (PRE) of target genes to alter the rate of transcription. However, the administration of progestins to endometrial cancer cells without PR expression does not have an inhibitory effect and could not be explained by the classical pathway of progesterone. Studies reported the existence of novel non-transcriptional mechanisms of signal transduction in hormone receptors. These so-called ’nongenomic’ effects do not depend on gene transcription or protein synthesis and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. These results provide evidence that the proliferation effect of progestins on progestin-resistant endometrial cancer cells might be mediated by a non-transcriptional pathway other than the PR classical pathway. Ubiquitous regulatory cascades such as mitogen-activated protein kinases, the phosphatidylinositol 3-OH kinase and tyrosine kinases are modulated through non-transcriptional mechanisms by steroid hormones, and it has recently been shown that Akt activation by growth factors such as insulin-like growth factor (IGF) induced endocrine resistance and downregulation of PR in metastatic breast cancer. Progestin might activate the PI3K/Akt pathway in endometrial cancers independent of PR.The study is to investigate the molecular events that lead to progestin resistance, the relationship between progestin resistance, growth factors activation and metabolism imbalance and to find a possible way to reverse progestin resistance in endometrial cancer. The cell lines are including ER and PR positive, progestin sensitive endometrial cancer cell lines, ER and PR low expression or negative, progestin resistant endometrial cancer cell lines and the progestin-resistant Ishikawa cell line which we established by long term progestin treatment.These experiments were divided into 3 parts, as following:PartⅠ. Differential effects of progestin on the PI3K/Akt pathway in progestin-sensitive and progestin-resistant endometrial cancer cells; PartⅡ. Inhibition of the PI3K/Akt pathway reversed progestin resistance in endometrial cancer. PartⅢ:Study on protein expression in progestin-resistance endometrial cancer cells by reverse phase protein array and metformin reverse progestin resistance. Part I.Differential effects of progestin on the PI3K/Akt pathway in progestin-sensitive and progestin-resistant endometrial cancer cellsObjective To investigate progestin effect to the PI3K/Akt pathway in endometrial carcinoma cells expressing different PR level expression and its relationship to progestin resistance.Methods Progestin-resistant cell line was developed from Ishikawa human endometrial adenocarcinoma cells by stepwise selection in increasing concentrations of the synthetic progestin, MPA (1—10μM). SRB Assay was used to detect the effect of MPA on the proliferation of the original Ishikawa cell line, the progestin—resistant subcell line and HEC-1A cell line. RT-PCR and Western blot were used to detect the expression of PR in mRNA and protein level. We knocked down the PR expression in the Ishikawa cells using siRNA and transfected pcDNA PR plasmid to overexpress PR in progestin-R Ishikawa cells and HEC-1A cells to address whether MPA activates Akt phosphorylation through PR.Results (1) MPA inhibited cell growth in a dose-dependent manner in the Ishikawa cells, and the growth inhibition rates were 17.8%,22.3% and 32.9% when treated with 1,10 and 20μM MPA, respectively, for 72 h. However, this inhibitory effect was not obviously in the progestin-R Ishikawa cells and the HEC-1A cells. (2) The MPA inhibited the PI3K/Akt pathway in progestin-sensitive Ishikawa cells, but activated the PI3K/Akt pathway in progestin-resistant Ishikawa cells or HEC-1A cells. (3) Progestin activated Akt phosphorylation in a PR-independent manner in the endometrial cancer cells with very low or no PR expression, but inhibited Akt phosphorylation in a PR-dependent manner in PR-overexpressed endometrial cancer cells.Conclusion Long-term progestin treatment downregulates PR expression in progestin-sensitive endometrial cancer cells and leads to progestin resistance. In addition, progestin could activate the PI3K/Akt pathway independent of PR mediation in PR-negative or low-expression endometrial cancer cells.Part II. Inhibition of the PI3K/Akt pathway reversed progestin resistance in endometrial cancerObjective To investigate the relationship between inhibiting PI3K/Akt pathway and reversing progestin resistance in endometrial cancer.Methods The PR expression in progestin-R Ishikawa cells was measured at 0,24,48,72 and 96 h of treatment with 10μM MPA in the presence of 1μM of LY294002. We used a general linear model to test the aititumor effect of MPA and/or LY294002 group. Endometrial cancer xenograft studies in nude mice was developed to detect the inhibiting effect of MPA and/or LY294002 in vivo.Results (1) Inhibition of the PI3K/Akt pathway restores PR expression in progestin-resistant cancer cells. (2) Inhibition of the PI3K/Akt pathway diminishes cell growth especially in progestin-resistant endometrial cancer cells:The combined treatment of 10μM MPA and 1μM LY294002,10μM MPA and 5μM LY294002 induced a further 33.7%,49.5%,48.1% and 44.5%,70.8%,60.9% reduction of the proliferation of Ishikawa, progestin-R Ishikawa and HEC-1A cells, respectively. (3) Inhibition of the PI3K/Akt pathway reversed progestin resistance in endometrial cancer in vivo, and the combination of LY294002 and MPA exhibited the most significant antitumor effects on both the progestin-sensitive and progestin-resistant endometrial cancer xenografts.:The tumor volume of Ishikawa, progestin-R Ishikawa and HEC-1A xenografts treated with LY294002 and MPA was significantly reduced by 44.9%,58.7% and 38.2%, respectively.Conclusions Inhibition of the PI3K/Akt pathway could restore PR expression and progestin sensitivity in progestin-resistant endometrial cancer. The combination treatment of progestin and PI3K inhibitor might be an effective conservative regimen for endometrial cancer patients. PartⅢ.Detecting protein expression in progestin-resistance endometrial cancer cells by RPPA and initial study on reversing progestin-resistance fucntion of metforminObjective To investigate the effect of metformin on endometrial cancer cells espically on progestin-resistant endometrial cancer cells, and its effect on PI3K/Akt pathway and PR clssical pathway.Methods Reverse phase protein array (RPPA) is used to detect and make comparison of 115 important genes in the proliferaton or apoptosis pathway in progestin-sensitive and -resistant endometrial cancer cells. Western blot is used to detect the IGF-1R and IR expression in progestin-sensitive and-resistant cells. SRB Assay was used to detect the effect of metformin on the proliferation of the endometrial cancer cell lines. Western blot is used to detect the effect of metformin on the change of AMPK, pAMPK and PR protein expression in endometrial cancer cells. RT-PCR is used to detect the downstream genes of PR in endometrial cancer cells after metformin stimulation.Results (1) IGF-1R pathway is overactivation in progestin-resistant endometrial cancer cells; (2) Metformin could restore progestin sensitivity in progestin-resistant endometrial cancer; (3) Restore progestin sensitivity by metformin is related to inhibiting PI3K/Akt pathway and upregulation PR transcription in progestin-resistant endometrial cancer cells.Conclusions Metformin could restore progestin sensitivity in progestin-resistant endometrial cancer, and its mechanisms are including activating AMPK, inhibiting PI3K/Akt and IGF-1R pathway and restore PR classical pathway in cells.Our results indicate that activation of the PI3K/Akt pathway by progestin without PR mediation plays an important role in progestin resistance to endometrial cancer cells. PR level should be taken as a crucial criterion by physicians in evaluating whether their patient needs progestin treatment. Long-term progestin treatment downregulates PR expression in progestin-sensitive endometrial cancer cells and leads to progestin resistance. Inhibition of the PI3K/Akt pathway could restore PR expression and progestin sensitivity in progestin-resistant endometrial cancer. Metformin could restore progestin sensitivity in progestin-resistant endometrial cancer, and its mechanisms are including activating AMPK, inhibiting PI3K/Akt and IGF-1R pathway and restore PR classical pathway in cells.In conclusion, the present study suggests that long-term progestin progestin could activate the PI3K/Akt pathway independent of PR mediation in PR-negative or low-expression endometrial cancer cells. Inhibition of the PI3K/Akt pathway could restore PR expression and progestin sensitivity in progestin-resistant endometrial cancer. There may exist the "AMPK-IGF-1R-PR/PI3K/Akt" network in endometrial cancer cells. The combination treatment of progestin and PI3K inhibitor, or metformin might be an effective conservative regimen for endometrial cancer patients who unfortunately developed progestin resistance but hoped to preserve their ability to remain fertile.