Aripiprazole as an Inhibitor of the EGFR/PI3K/AKT Signaling Pathway and Sensitizer of MPA Suppressed Progestin-Resistant Endometrial Cancer Cells.

Role of glucocorticoid receptor in the inhibitory effect of medroxyprogesterone acetate on the estrogen-induced endothelial nitric oxide synthase phosphorylation in human umbilical vein endothelial cells

BackgroundProgestin is effective to promote endometrial cancer (EC) cells apoptosis, however, continuous progestin administration causes low level of progestin receptor B (PRB), further resulting in progestin resistance. Here, we performed microarray analysis on Ishikawa cells (PRB+) treated with medroxyprogesterone acetate (MPA) to explore the molecular mechanism underlying the inhibitory influence of MPA on PRB+ EC cells.MethodsMicroarray analysis was performed by using Ishikawa cells (PRB+) treated with MPA. Differentially expressed mRNA and long noncoding RNAs (lncRNAs) were identified. Furthermore, the functions of these mRNAs and lncRNAs were predicted by functional enrichment analysis. QRT-PCR was further performed to verify the microarray data.ResultsA total of 358 differentially expressed genes and 292 lncRNAs were identified in Ishikawa cells (PRB+) treated with MPA. QRT-PCR verified these data. Functional enrichment analysis identified endoplasmic reticulum (ER) stress as the key pathway involved in the inhibitory effect of MPA on EC cells. And the ER stress apoptotic molecule CHOP and ER stress related molecule HERPUD1 were both highly expressed in Ishikawa cells (PRB+) treated with MPA. Co-expression analysis showed lnc-CETP-3 was highly correlated with CHOP and HERPUD1, suggesting it might participate in ER stress pathway-related EC cell apoptosis caused by MPA. In addition, compared with untreated cells, lnc-CETP-3, CHOP and HERPUD1 were significantly up-regulated in Ishikawa cells (PRB+) treated with MPA, whereas they have no statistical significance in KLE cells (PRB-).ConclusionsMPA may activate ER stress by progesterone-PRB pathway to up-regulate CHOP expression, which may be one of the molecular mechanisms underlying the inhibitory effect of MPA on EC cells with PRB+. Lnc-CETP-3 might be involved in this process. These findings may provide therapeutic targets for EC patients with PRB-, and resistance-related targets to increase the sensitivity of MPA on EC cells.

The aim of these investigations was to study the role of gefitinib (a specific oral epidermal growth factor receptor-tyrosine kinase inhibitor) on reversing progestin-resistance in a human endometrial carcinoma xenograft model. To study the effect of gefitinib and epidermal growth factor receptor (EGFR) overexpression on tumor progestin resistance, the Ishikawa endometrial carcinoma cell line was transfected to stably express a high level of EGFR, which resulted in the progestin-resistant Ishikawa-pLWERNL subcell line. BALB/c nude mice were injected subcutaneously with the parental Ishikawa cell line and the Ishikawa-pLWERNL cell line. Therapy experiments with gefitinib alone or in combination with medroxyprogesterone acetate (MPA) were done and samples were analyzed for EGFR and progesterone receptor isoform B (PR-B) expression by Western blot and immumohistochemistry analyses. Role in blocking EGFR autophosphorylation and its downstream signaling pathway and antagonizing progestin resistance by gefitinib was investigated by Western blot analysis. EGFR expression was higher in progestin-resistant Ishikawa-pLWERNL endometrial cancer (EC) xenografts than in progestin-sensitive Ishikawa EC xenografts; in contrast, PR-B was higher in Ishikawa xenografts than in Ishikawa-pLWERNL xenografts. Higher EGFR expression reduced sensitivity to progestin and decreased PR-B expression in Ishikawa xenografts; it also abnormally activated EGFR autophosphorylation and its downstream signaling pathway. Gefitinib effectively inhibited the proliferation of EC xenografts that overexpressed EGFR, and reversed hormone resistance in progestin-resistant EC xenografts. The present study describes an in vivo model that can provide a valuable tool in studying the interaction of overexpressed EGFR and progestin resistance in EC. Gefitinib may be useful in the treatment of progestin-resistant EC.

Progestin is one of the main hormone treatment regimens for early-stage estrogen receptor- and progesterone receptor (PR)-positive endometrial cancer (EC). However, the response rate of EC to progestins is unsatisfactory. Investigating the mechanisms related to progestin treatment could help improve treatment efficacy. Studies have demonstrated that normal endometrial stromal cells (ESCs) increase the inhibitory effect of progestin on EC cell proliferation via paracrine signaling, but the mechanisms involved remain unclear. In this study, we found that ESCs had different morphological features between progestin-sensitive and -insensitive EC tissues. ESCs presented typical decidualization changes in progestin-sensitive cases, while they remained slim in progestin-insensitive EC lesions, indicating no response. Furthermore, ESCs enhanced the inhibitory effect of medroxyprogesterone acetate (MPA) on EC cell proliferation by secreting neuron cell adhesion molecule (NrCAM). MPA treatment enhanced NrCAM secretion by ESCs. EC xenografts in BALB/C nude mice demonstrated that MPA combined with NrCAM had an increased tumor inhibitory effect compared with MPA or NrCAM alone. Mechanistically, MPA upregulated NrCAM expression in ESCs through PR. Specifically, NrCAM increased PR expression in EC cells through TET1-induced hydroxymethylation of the PRB gene promoter region. These findings indicate that NrCAM or NrCAM combined with progestins could be a new EC treatment.

Fertility-sparing management is an important treatment for young women who have endometrial carcinoma (EC). Many patients with EC exhibit insensitivity or resistance to progestin therapy, and the molecular mechanisms of that insensitivity and resistance have been elusive. Epidermal growth factor receptor (EGFR) overexpression has been observed in EC, but the roles of EGFR in progestin resistance have not been investigated. EGFR and progesterone receptor isotype B (PR-B) messenger RNA and protein levels were determined in EC tissue samples and cell lines by immunohistochemical, real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. The biologic function of EGFR in the regulation of PR-B expression and induced progestin resistance was investigated by transfecting recombinant plasmids that expressed EGFR into Ishikawa EC cells. In addition, the role of the mitogen-activated protein kinase (MAPK) signal pathway was investigated by Western blot analysis. EGFR was detected in 60% of PR-B-positive samples and in 90.5% of PR-B-negative samples (P=.015). EGFR expression was higher in progestin-resistant KLE EC cells than in progestin-sensitive Ishikawa EC cells. In contrast, PR-B expression was higher in Ishikawa cells than KLE cells. Higher EGFR expression reduced sensitivity to progestin and decreased PR-B expression in Ishikawa cells; it also abnormally activated the MAPK signaling pathway and inhibited cell apoptosis. The EGFR tyrosine kinase-specific inhibitor AG1478 effectively inhibited the proliferation of EC cells that overexpressed EGFR. The current results indicated that EGFR overexpression may play an important role in reducing sensitivity to progestin treatment in EC. An EGFR tyrosine kinase-specific inhibitor may be useful in the treatment of EC.

To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces EGFR transactivation through HB-EGF ectodomain shedding. EGFR and its downstream ERK and PI3K signaling pathways play a role in preventing epithelial apoptosis in the early stage of bacterial infection.

EGF receptor activation by G-protein coupled receptors

Inhibitors of the epidermal growth factor receptor (EGFR) have generated considerable hope for cancer treatment, specifically for lung and breast cancers. Therefore, identification of a natural, nontoxic agent(s) as an inhibitor of EGFR is of considerable importance. Delphinidin, an anthocyanidin present in pigmented fruits and vegetables, possesses potent antioxidant and antiproliferative properties. In our study, employing EGFR positive breast cancer AU-565 cells and immortalized MCF-10A cells, we evaluated the effect of delphinidin on EGFR and its downstream signaling pathways. Delphinidin (5-40 microM; 3 hr) treatment of both AU-565 cells and MCF-10A cells inhibited the (i) phosphorylation of EGFR, (ii) activation of PI3K, (iii) phosphorylation of AKT and MAPK. Further, delphinidin treatment of AU-565 cells inhibited EGF-induced autophosphorylation of EGFR, AKT and MAPK, activation of PI3K and cell invasion. We then compared the growth inhibitory effects of delphinidin (5-40 microM; 48 hr), and found that it resulted in a decrease in cell growth of AU-565 and MCF-10A cells but had only minimal effects on normal mammary epithelial 184A1 cells. Treatment of AU-565 cells with delphinidin resulted in (i) induction of apoptosis, (ii) cleavage of PARP protein, (iii) activation of caspase-3 and (iv) downregulation of Bcl-2 with an increase in the expression of Bax. In summary, our study identifies a naturally occurring dietary agent delphinidin as an effective inhibitor of EGFR signaling in breast cancer cells. We suggest that delphinidin could be developed as an agent for the management of EGFR positive human cancers.

Epidermal Growth Factor Receptor Is a Critical Mediator of Ultraviolet B Irradiation-Induced Signal Transduction in Immortalized Human Keratinocyte HaCaT Cells

To explore the in vitro effects of thioridazine (THIO) plus medroxyprogesterone (MPA) on the proliferation and apoptosis of endometrial cancer cell lines (Ishikawa & KLE) and examine the mechanism in the treatment of endometrial cancer. CCK-8 assay was employed for detecting the influence of THIO plus MPA on the proliferation and apoptosis of endometrial cancer cells (ISK & KLE). And the concentration and timepoints of drugs were determined according to the results. Real-time polymerase chain reaction (PCR) and Western blot were used to detect the expression levels of PRB, DRD2 and p-AKT/AKT in PI3K/AKT signal pathway. Flow cytometry was applied for detecting the apoptosis of combination (THIO+MPA) and MPA groups. Compared to MPA group, the proliferation inhibiting effect of combination group increased significantly in ISK and KLE cells (52.5% ± 2.6% vs 37.3% ± 0.3%, P < 0.05; 97.7% ± 0.7% vs 50.0% ± 0.4%, P < 0.001); the apoptotic rates increased (34.0% ± 1.4% vs 50.5% ± 2.4%, P < 0.01; 5.5% ± 3.6% vs 11.3% ± 0.7%, P < 0.01); the expression levels of PRB and DRD2 were up-regulated (all P < 0.05). And the ratio of p-AKT/AKT decreased obviously. Thioridazine significantly enhances the effects of MPA on proliferative inhibition and apoptotic promotion in endometrial cancer cells. And it may be associated with the PRB/DRD2-mediated PI3K/AKT signal pathway.

BackgroundPrecise prediction of drug combination targeting tumor cells effectively is a crucial challenge for tumor therapy, especially for endometrial cancer (EC). Considering the resistance, crosstalk that occurs between the receptor tyrosine kinase mesenchymal-epithelial transition factor (cMet) and epidermal growth factor receptor (EGFR), and their indispensable influence on the occurrence of EC, this study aimed to explore a novel therapeutic approach for EC treatment through blocking cMet and EGFR simultaneously.MethodsIn the present study, the expression of miR-26a-5p in EC cell lines was detected using quantitative real-time polymerase chain reaction assay. The potential role of miR-26a-5p in the development of EC was examined using cell counting kit assay, 5-ethynyl-2’- deoxyuridine staining, wound healing assay, and cell apoptosis staining assay. Subsequently, the effect of upregulated miR-26a-5p in vivo was confirmed on a xenograft model. Luciferase reporter assay and Western blot analysis were performed to verify the relation between miR-26a-5p and cMet. Furthermore, the dual therapeutic effect of miR-26a-5p and EGFR monoclonal antibody cetuximab was confirmed in vivo and in vitro.ResultsThe results indicated that miR-26a-5p expression significantly reduced in EC cell lines compared with the normal endometrial cell line. Furthermore, the overexpression of miR-26a-5p inhibited the progression of EC, including cell migration, cell proliferation, and cell apoptosis in vivo and in vitro. Subsequently, mir-26a-5p regulated the expression of cMet and the downstream the hepatocyte growth factor (HGF)/cMet pathway, thus exerting an inhibitory effect on EC cells. In addition, the study also demonstrated that the upregulation of miR-26a-5p could significantly enhance the inhibitory effect of cetuximab compared with the use of cetuximab alone in vivo and in vitro.ConclusionsRNAi therapeutic miR-26a-5p suppressed the progression of EC through regulating the cMet/HGF pathway. The dual therapy using RNA interference and neutralizing antibody simultaneously blocked tumor targets, including cMet and EGFR, thus providing a novel approach for overcoming the resistance to the inhibitors against a single target in EC treatment.

Members of the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases have been implicated in the pathogenesis of various malignancies. The ability of one EGFR subfamily member to influence, or function synergistically with, another is likely to be a general feature of these receptors. To assess the role of receptor heterodimerization, we analyzed the ability of Neu differentiation factor (NDF) to induce cell growth and transformation of NIH 3T3 cells transfected with different combinations of the EGFR subfamily of receptors. NDF induced mitogenesis, but not transformation, of cells expressing either HER3 or HER4 alone. However, NDF-induced cell transformation was observed when either HER1 or HER2 was coexpressed with HER3 or HER4. In analogous receptor phosphorylation experiments, NDF-induced transphosphorylation appears to be correlated with synergistic transformation of NIH 3T3 cells. Interestingly, transphosphorylation between HER1 and HER4 can be stimulated by either EGF or NDF.

The murine retroviral oncogene v-cbl induces pre-B cell lymphomas and myelogenous leukemias. The protein product of the mammalian c-cbl proto-oncogene is a widely expressed cytoplasmic 120-kDa protein (p120cbl) whose normal cellular function has not been determined. Here we show that upon stimulation of human epidermal growth factor (EGF) receptor, p12ocbl becomes strongly tyrosine-phosphorylated and associates with activated EGF receptor in vivo. A GST fusion protein containing amino acids 1-486 of p120cbl, including a region highly conserved in nematodes, binds directly to the autophosphorylated carboxyl-terminal tail of the EGF receptor. Platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or nerve growth factor (NGF) stimulation also results in tyrosine phosphorylation of p120cbl. Recent genetic studies in Caenorhabditis elegans indicate that Sli-1, a p120cbl homologue, plays a negative regulatory role in control of the Ras signaling pathway initiated by the C. elegans EGF receptor homologue. Our results indicate that p120cbl is involved in an early step in the EGF signaling pathway that is conserved from nematodes to mammals.

Objective To study the differential expression of epidermal growth factor receptor(EGFR),phosphoinositide 3 kinase(PI3K) and its phosphorylated protein p-PI3K,and to analyze the correlation between the expression of the three proteins and clinicopathological features in different subtypes of breast cancer,and to investigate the role of PI3K and its phosphorylated protein p-PI3K in the PI3K/Akt/mTOR signaling pathway in the development and progression of breast cancer.Methods 117 cases of newly diagnosed invasive breast cancer were selected from Jan.2005 to Jun.2010 in Affiliated Cancer Hospital of Xinjiang Medical University,and molecular typing was made by immunohistochemistry results of ER,PR and HER-2,including triple negative breast cancer group(TN group,28 cases),HER-2 over-expression breast cancer group(HER-2 + group,12 cases)and luminal epithelial breast cancer group (Luminal group,77 cases).Immunohistochemistry was used to detect the expressions of PI3 K,p-PI3 K and EGFR in breast carcinoma and its adjacent tissues,and to analyze their expression characteristics in breast cancer and its different subtypes,as well as the correlations between their expressions and clinicopathological features.Results (1) Expressions of PI3K,p-PI3K and EGFR in breast cancer tissues and the adjacent tissues were different:the positive expression rates of PI3K and p-PI3K in breast cancer tissues were higher those that in the adjacent tissues,and the difference had statistical significance (x2 =27.589,P ＜ 0.001 ;x2 =49.327,P ＜ 0.001).EGFR expressions were significantly different in breast cancer tissues and the adjacent tissues(x2 =10.920,P =0.004).(2) Expression of PI3K,p-PI3K and EGFR in different subtypes of breast cancer was different:the positive expression rates of PI3k and p-PI3K were similar between TN group and HER-2 + group,but lower than those in Luminal group,while the expression of EGFR in TN group was significantly higher than those in the other two groups.Expressions of PI3K,p-PI3K and EGFR were statistically different in different subtypes of breast cancer (x2 =9.842,P =0.037 ;x2 =13.423,P =0.006 ;x2 =12.410,P =0.012).Likewise,expressions of PI3K,p-PI3K and EGFR were different between the three different groups (x2 =7.835,P =0.020;x2 =11.791,P =0.003).Expressions of PI3K and EGFR were statistically different only in TN group and Luminal group.There were statistically difference in p-PI3K expression between TN group and Luminal group,and between HER-2 + group and Luminal group(x2 =9.336,P =0.009 ;x2 =7.361,P =0.025).There was no significant difference in the other groups.(3)The correlations of the expressions of PI3 K and p-PI3 K in breast cancer and three different subtypes:compared with PI3K,the positive expression rate of p-PI3K significantly increased in breast cancer,and showed significant positive correlation(r =0.324,P ＜ 0.001).However,this correlation was different between the three different groups.Expressions of PI3k and p-PI3K showed a positive correlation in Luminal group (r =0.385,P =0.001),and there was no relation in TN group and HER-2 group (r =0.222,P =0.257 ; r =0.410,P =0.185).Further stratified analysis showed that the expression status of EGFR affected the expressions of PI3K and p-PI3K in breast cancer.No significant correlation was found in the EGFR negative group(r =0.186,P =0.227),while there was significant positive correlation in EGFR positive group(r =0.396,P =0.001).(4)Relations between expressions of PI3K and p-PI3K and clinicopathological features of breast cancer:there were differences in expressions of PI3K between different ethnic Han and Uighur,as well as between different histological grades (x2 =7.846,P =0.020 ;x2 =7.160,P =0.028),and no correlation with tumor size,axillary lymph node status and tumor staging was found.The expression of p-PI3 K was not related to the clinicopathological features in breast cancer.Conclusions EGFR may play a role in carcinogenesis of breast cancer through activation of the PI3K,and the expression status of EGFR affects the activation of PI3 K signaling pathway.After stimulated by the upstream impact factors,PI3K can be activated and play an important role in the signal transduction pathway.The expressions of PI3K and its phosphorylated proteins p-PI3K are different in different subtypes of breast cancer.The results suggest that individual treatment should be made according to different types of breast cancer,and PI3K may become a new target for breast cancer treatment. Key words: Breast cancer; Molecular subtypes; PI3K signaling pathway; Phosphorylation; Epidermal growth factor receptor

Phase II study of medroxyprogesterone acetate plus metformin as a fertility-sparing treatment for atypical endometrial hyperplasia and endometrial cancer