First trimester human placental factors induce breast cancer cell autophagy.

Placental factors, progesterone included, facilitate breast cancer cell line (BCCL) motility and thus may contribute to the advanced breast cancer found during pregnancy. Cancer and placental implantations are similar; the last is accompanied by extravillous trophoblast cell invasion and autophagy which are interlinked. We aimed to analyze the effect of first trimester human placenta on BCCL autophagy. BCCLs (MCF-7/T47D) were cultured with placental explants (60 h) or placental supernatants (24 h). Following cultures, BCCLs were sorted out for RNA/protein extraction. RNA served for microarray/qPCR (BNIP3) and protein for Western blot (HIF1α, LC3BII) analyses. Inhibitors were added to the placenta-MCF-7 coculture or placental supernatants (autophagy inhibitor-3MA, progesterone receptor (PR) inhibitor-RU486, and HIF1α inhibitor-Vitexin) in order to evaluate their effects on BCCL motility and LC3BII/HIF1α expression. LC3BII (an autophagy marker) expression was elevated in BCCLs following placental explant coculture and exposure to placental supernatants. The autophagy inhibitor (3MA) repressed the placenta-induced MCF-7/T47D migration, establishing a connection between BCCL autophagy and migration. Microarray analysis of MCF-7 following placenta-MCF-7 coculture showed that "HIF1α pathway," a known autophagy facilitator, was significantly manipulated. Indeed, placental factors elevated HIF1α and its target BNIP3 in the BCCLs, verifying array results. Lastly, PR inhibitor reduced HIF1α expression and both PR and HIF1α inhibitors reduced MCF-7 LC3BII expression and motility, suggesting involvement of the PR-HIF1α axis in the autophagy process. Placental factors induced BCCL autophagy that is interlinked to their motility. This suggests that autophagy-related molecules may serve as targets for therapy in pregnancy-associated breast cancer.

The effect of heat shock protein 27 on extravillous trophoblast differentiation and on eukaryotic translation initiation factor 4E expression.

Heat shock protein (HSP27) is expressed in human placentae. Previously, we showed that HSP27 is expressed in the villous cell column of first trimester placental explants and in extravillous trophoblast (EVT) cells. EVT differentiation is accompanied by increased motility, matrix metalloproteinase (MMP) activity, decreased proliferation and expression of specific markers such as HLAG and CD9. HSP27 regulates cell apoptosis, migration, protein stability and the availability of eukaryotic translation initiation factors, such as eukaryotic translation initiation factor 4E (eIF4E). eIF4E supports trophoblast cell proliferation and survival. We wanted to explore the effect of HSP27 silencing on trophoblast cell phenotype, EVT markers and eIF4E expression and regulators [4E-binding protein (4E-BP1) and MAP kinase-interacting kinase (MNK1)]. This study evaluated the effect of HSP27 siRNA on placental explant and HTR-8/SVneo migration, MMP activity/mRNA, cell death, cell cycle, HLAG/CD9 levels, and eIF4E and its regulators' total and phosphorylated levels. Furthermore, we evaluated HSP27 levels in placentae exposed to ribavirin, which triggers EVT differentiation. We found that HSP27 silencing increased cell death in HTR-8/SVneo and placental explants. Furthermore, it reduced HTR-8/SVneo migration and EVT outgrowth from the explants (P < 0.05), MMP2 activity and expression of EVT markers HLAG and CD9 (in placental explants and HTR-8/SVneo, respectively, P < 0.05). Induction of EVT differentiation by ribavirin elevated HSP27 levels. Finally, HSP27 silencing in both HTR-8/SVneo and placental explants reduced eIF4E levels (33 and 28%, respectively, P < 0.05) and the levels of its regulators 4E-BP1 and MNK1 (37 and 32%, respectively, done on HTR-8/SVneo only), but not their phosphorylated forms. Altogether, our results suggest that HSP27 contributes to EVT cell differentiation.

The involvement of eukaryotic translation initiation factor 4E in extravillous trophoblast cell function.

UNLABELLED: Extravillous trophoblast cells (EVT) are major players in placental implantation. They differentiate in the villous cell column, invade to the uterus and remodel the uterine spiral arteries. Trophoblast and tumor cells have similar invasion mechanisms, share similar biochemical mediators (e.g. c-myc, MMP9) and growth-factors (e.g. VEGF). The mRNA of these proteins has extremely structured 5-UTR and their translation is highly dependent on eukaryotic-translation-initiation-factor-4E (eIF4E). Cancer cells have elevated eIF4E and are more vulnerable to its silencing than normal cells. We speculated that like cancer, trophoblast function is highly eIF4E dependent. OBJECTIVE: Analyze eIF4E involvement in EVT differentiation and function. STUDY DESIGN: EIF4E levels were assessed in first-trimester human placentae and in placental explants before and after EVT differentiation. The effect of eIF4E knockdown (siRNA, ribavirin) on the phenotype of placental explant and EVT cell lines (HTR-8/SVNEO) was evaluated. Tested parameters included eIF4E and its target levels, migration, invasion, cell death, cell cycle and cell count. RESULTS: High eIF4E levels were found in cytotrophoblast and especially EVT cells during their differentiation in the villi, compared to other placental cell types. EIF4E silencing increased cell death and cell cycle arrest in placental explants and HTR-8/SVNEO cells. Although it induced EVT outgrowth in the placental explants, it reduced HTR-8/SVNEO motility, reflecting the importance of using ex vivo models that include an intact placental microenvironment in its original architecture. CONCLUSIONS: Our results suggest that eIF4E prevents final EVT differentiation and supports placental cell proliferation and survival. A balance between cell proliferation and differentiation is crucial for placental development and implantation.

Hormone-dependent placental manipulation of breast cancer cell migration.

BACKGROUND: Breast cancer during pregnancy is often more advanced than in non-pregnant women. Nevertheless, no case of metastasis inside the placenta has been reported. Previously, we showed that placental-explants eliminated breast cancer cells from their surroundings, due to cell-death and elevated migration. Our objective was to find the underlying mechanisms of these phenomena. METHODS AND RESULTS: Our model contained Michigan Cancer Foundation 7 (MCF7) or T47D cells co-cultured with and without human placental explants. Microarray analysis, validated by quantitative PCR, of MCF7 following their placental co-culture suggested activation of estrogen (E(2)) signaling. As extensive cross-talk exists between E(2) and progesterone, their involvement in mediating placental effects on breast cancer cells was tested. Indeed, addition of E(2) and progesterone receptor (ER and PR) inhibitors to the co-culture system reduced cancer cell motility, yet did not alter cell-cycle or death. E(2) and progesterone concentrations in placental media were found to be similar to those of early pregnancy blood levels. Interestingly, placental-breast cancer co-culture media contained lower progesterone (P < 0.05) and higher E(2) (200%, P < 0.05) levels than placentae cultured separately. Placental supernatant and E(2) and progesterone at placental levels were sufficient to increase MCF7 and T47D migration and invasion (P < 0.05), yet did not alter MCF7 cell-cycle or death. Furthermore, placental supernatant elevated p38 and Jun N-terminal kinase (JNK) phosphorylation in both cell lines (P < 0.05). Inhibitors of JNK, ER and PR reversed MCF7 and T47D motility induced by the placenta, suggesting their involvement. CONCLUSIONS: We suggest that E(2) and progesterone contribute to cell migration away from placental areas. We hypothesize that they may increase metastatic spread to other organs in pregnancy.