Functions And Mechanism Of Estrogen Combined Shh/Gli Signaling On Breast Cancer Stem Cells And Epithelial-mesenchymal Transition

ObjectiveTo explore the role of estrogen and Sonic hedgehog (Shh)/Gli signaling pathway on breast cancer stem cells (BCSCs) and intercellular epithelial-mesenchymal transition (EMT); to research the effect of estrogen on Shh/Gli signaling pathway, BCSCs characteristics and EMT; to observe the change of BCSCs and EMT by blocking shh/Gli pathway; to explore the pathogenesis and new targeted therapies of breast cancer.MethodsEndogenous mRNA expression of breast cancer stem cell markers ALDH1, Gli1(a Hedgehog target gene), and estrogen receptor (ER) in several human breast cancer cell lines were determined by reverse transcription polymerase chain reaction (RT-PCR). Then, the relationships among Gli1, ALDH1, and ER expression levels were analyzed by linear correlation assay. The expression of ER protein was measured by western blotting and immunofluorescence assays in MCF-7, HCC1428, MDA-MB-231, and BT549cells. The four cell lines were incubated with10nM estrogen (E2) with or without1μM4-hydroxy tamoxifen (4OHT) for4days, then Gli1protein and mRNA expression were measured by western blotting and RT-PCR. MCF-7and HCC1428cells were transfected with pSingle vectors carrying short hairpin RNA (shRNA) targeting Gli1. Flow cytometry, mammosphere formation and western blot were used to detect the influence in BCSCs; wound scratch, Matrigel invasion assay and Western blot were used to measure EMT. MCF7cells were treated with E2(0-10nM) for4days. Gli1, Shh, ALDH1, Nanog, SOX2, and Bmi-1expression levels were measured by RT-PCR and western blotting. The tissue microarray containing100breast cancer samples and10adjacent normal breast tissues or adenosis samples to analyze the expression of ER, Gli1, and ALDH1by immunohistochemical staining. Then, the relationships among ER, Gli1, and ALDH1expression were analyzed by linear correlation assay. Results1. ALDH1and Glil mRNAs were detectable in all cell lines, and ER expression was positively correlated with Gli1and ALDH1(r=0.845,p<0.01; r=0.385,p<0.05).2. In MCF-7and HCC1428cells, Gli1expression was significantly increased in estrogen-treated cells compared with that in control (EtOH-treated) cells. The estrogen-induced activation of Gli1was inhibited by4OHT (p<0.01). However, E2failed to significantly increase Gli1expression in MDA-MB-231and BT549cells. The results indicated that estrogen activated the Shh/Glil pathway only in ER-positive breast cancer cells through Shh/Gli signaling.3. In E2induced MCF-7and HCC1428shVEC cells, CD44+/CD24-/low stem-like cells were statistically significant expansion compared with EtOH-treated cells, but in Gli1-knockdown cells (shGli1-1or shGlil-2), E2failed to significantly increase the proportion of CD44+/CD24-/low stem-like cells. Compared with shVEC cells, the proportion of CD44+/CD24-/low stem-like cells of shGlil-1or shGli1-2cells were decreased. The similar results were obtained from mammosphere formation. In E2induced shVEC cells, the number and size of these spheres were statistically significant increased. In shGli1-1or shGlil-2cells, estrogen failed to significantly increase the number and size of these spheres. The results indicated that Glil was indeed involved in the regulation of estrogen-induced self-renewal of breast CSCs (**,##P<0.01).4. Glil, Shh, ALDH1, Nanog, SOX2, and Bmi-1were all gradually increased with the increase in E2concentration, whereas Shh was nearly unchanged. We hypothesized that estrogen contributed to increased numbers of breast CSCs through a noncanonical ligand-independent Shh/Gli signaling pathway (*p<0.05,**P<0.01).5. The abilities of migration and invasion of estrogen induced shVEC cells were significantly enhanced; compared with shVEC, migration and invasion of shGli1-1or shGlil-2cells decreased; shGlil-1or shGlil-2cells estrogen-treated group and alcohol control group migration and invasion of little change. These data indicated that Gli1plays a significant role in mediating the invasiveness of ER-positive breast cancer cells (**,##P<0.01). 6. Glil and ALDH1protein expression levels of tissue microarray were low or undetectable in the10normal samples, but quite high in breast tumor tissues. Moreover, ER expression was positively correlated with Glil and ALDH1expression levels.ConclusionEstrogen may act via Gli1to promote stem cell development and the EMT in ER-positive breast cancer cells, which may contribute to breast tumor malignancy. Thus, Glil may be potential therapeutic targets to mediate cancer stem cells in the development of novel treatments for breast cancer.