Differentiation of first trimester human placental cytotrophoblast (CTB) from an anchorage-dependent epithelial phenotype into the mesenchymal-like invasive extravillous trophoblast (EVT) is crucial in the development of the maternal–fetal interface. We showed previously that differentiation of first trimester CTB to EVT involves an epithelial-mesenchymal transition (EMT). Here we compare the epithelial-mesenchymal characteristics of CTB and EVT derived from normal third trimester placenta or placenta previa versus abnormally invasive placenta (AIP). CTB and EVT were isolated from normal term placenta or placenta previa following Caesarean section and EVT from AIP following Caesarean hysterectomy. Cell identity was validated by measurement of cytokeratin-7 and HLA-G. Comparing normal term CTB with EVT from normal term placenta or placenta previa for differential expression analysis of genes associated with the EMT showed changes in >70% of the genes probed. While demonstrating a mesenchymal phenotype relative to CTB, many of the gene expression changes in third trimester EVT were reduced relative to the first trimester EVT. We suggest that third trimester EVT are in a more constrained, metastable state compared to first trimester equivalents. By contrast, EVT from AIP demonstrate characteristics that are more mesenchymal than normal third trimester EVT, placing them closer to first trimester EVT on the EMT spectrum, consistent with a more invasive phenotype.

Recent studies suggest that peroxisome proliferator‐activated receptor gamma (PPARγ) agonists may have cancer chemopreventive activity. Other studies have shown that loss of epidermal PPARγ results in enhanced chemical carcinogenesis in mice via unknown mechanisms. However, ultraviolet B (UVB) exposure represents the primary etiological agent for skin cancer formation and the role of PPARγ in photobiology and photocarcinogenesis is unknown. In previous studies, we demonstrated that UVB irradiation of cells results in the formation of oxidized glycerophosphocholines that exhibit PPARγ ligand activity. We therefore hypothesized that PPARγ would prove to be a chemopreventive target in photocarcinogenesis. We first showed that UVB irradiation of mouse skin causes generation of PPARγ agonist species in vivo. We then generated SKH‐1 hairless, albino mice deficient in epidermal Pparg (Pparg−/−^epi) using a cytokeratin 14 driven Cre‐LoxP strategy. Using a chronic model of UVB photocarcinogenesis, we next showed that Pparg−/−^epi mice exhibit an earlier onset of tumor formation, increased tumor burden and tumor progression. Increased tumor burden in Pparg−/−^epi mice was accompanied by a significant increase in epidermal hyperplasia and p53 positive epidermal cells in surrounding skin lacking tumors. After acute UVB irradiation, Pparg−/−^epi mice exhibited an augmentation of both UVB‐induced Caspase 3/7 activity and inflammation. Increased apoptosis and inflammation was also observed after treatment with the PPARγ antagonist GW9662. With chronic UVB irradiation, Pparg−/−^epi mice exhibited a sustained increase in erythema and transepidermal water loss relative to wildtype littermates. This suggests that PPARγ agonists could have possible chemopreventive activity in non‐melanoma skin cancer.

Does the upregulation of the zinc finger E-box binding homeobox 2 (ZEB2) transcription factor in human trophoblast cells lead to alterations in gene expression consistent with an epithelial–mesenchymal transition (EMT) and a consequent increase in invasiveness? Overexpression of ZEB2 results in an epithelial–mesenchymal shift in gene expression accompanied by a substantial increase in the invasive capacity of human trophoblast cells. In-vivo results have shown that cytotrophoblast differentiation into extravillous trophoblast involves an epithelial–mesenchymal transition. The only EMT master regulatory factor which shows changes consistent with extravillous trophoblast EMT status and invasive capacity is the ZEB2 transcription factor. This study is a mechanistic investigation of the role of ZEB2 in trophoblast differentiation. We generated stable ZEB2 overexpression clones using the epithelial BeWo and JEG3 choriocarcinoma lines. Using these clones, we investigated the effects of ZEB2 overexpression on the expression of EMT-associated genes and proteins, cell morphology and invasive capability. We used lentiviral transduction to overexpress ZEB2 in BeWo and JEG3 cells. Stable clones were selected based on ZEB2 expression and morphology. A PCR array of EMT-associated genes was used to probe gene expression. Protein measurements were performed by western blotting. Gain-of-function was assessed by quantitatively measuring cell invasion rates using a Transwell assay, a 3D bioprinted placenta model and the xCelligence^TM platform. The four selected clones (2 × BeWo, 2 × JEG3, based on ZEB2 expression and morphology) all showed gene expression changes indicative of an EMT. The two clones (1 × BeWo, 1 × JEG3) showing >40-fold increase in ZEB2 expression also displayed increased ZEB2 protein; the others, with increases in ZEB2 expression <14-fold did not. The two high ZEB2-expressing clones demonstrated robust increases in invasive capacity, as assessed by three types of invasion assay. These data identify ZEB2-mediated transcription as a key mechanism transforming the epithelial-like trophoblast into cells with a mesenchymal, invasive phenotype. PCR array data have been deposited in the GEO database under accession number GSE116532. These are in-vitro studies using choriocarcinoma cells and so the results should be interpreted in view of these limitations. Nevertheless, the data are consistent with in-vivo findings and are replicated in two different cell lines. The combination of these data with the in-vivo findings clearly identify ZEB2-mediated EMT as the mechanism for cytotrophoblast differentiation into extravillous trophoblast. Having characterized these cellular mechanisms, it will now be possible to identify the intracellular and extracellular regulatory components which control ZEB2 and trophoblast differentiation. It will also be possible to identify the aberrant factors which alter differentiation in invasive pathologies such as preeclampsia and abnormally invasive placenta (AKA accreta, increta, percreta). Funding was provided by the Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery at Hackensack Meridian Health, Hackensack, NJ. The 3D bioprinted placental model work done in Drs Kim and Fisher’s labs was supported by the Children’s National Medical Center. The xCELLigence work done in Dr Birge’s lab was supported by NIH CA165077. The authors declare no competing interests.

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr −/− mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3 ^EGFP transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.

Field cancerization refers to areas of grossly normal epithelium that exhibit increased risk for tumor occurrence. Unfortunately, elucidation of the locoregional changes that contribute to increased tumor risk is difficult due to the inability to visualize the field. In this study, we use a noninvasive optical-based imaging approach to detail spatiotemporal changes in subclinical hyperemia that occur during experimental cutaneous carcinogenesis. After acute inflammation from 10 weeks of UVB irradiation subsides, small areas of focal hyperemia form and were seen to persist and expand long after cessation of UVB irradiation. We show that these persistent early hyperemic foci reliably predict sites of angiogenesis and overlying tumor formation. More than 96% of the tumors (57 of 59) that developed following UVB or 7,12-dimethylbenz(a)anthracene/phorbol 12-myristate 13-acetate (DMBA/PMA) treatment developed in sites of preexisting hyperemic foci. Hyperemic foci were multifocal and heterogeneously distributed and represented a minor fraction of the carcinogen-treated skin surface (10.3% of the imaging area in vehicle-treated animals). Finally, we also assessed the ability of the anti-inflammatory agent, celecoxib, to suppress hyperemia formation during photocarcinogenesis. The chemopreventive activity of celecoxib was shown to correlate with its ability to reduce the area of skin that exhibit these hyperemic foci, reducing the area of imaged skin containing hyperemic foci by 49.1%. Thus, we propose that a hyperemic switch can be exploited to visualize the cancerization field very early in the course of cutaneous carcinogenesis and provides insight into the chemopreventive activity of the anti-inflammatory agent celecoxib. Cancer Res; 73(1); 150–9. ©2012 AACR.