Spontaneous Transformation of Murine Oviductal Epithelial Cells: A Model System to Investigate the Onset of Fallopian-Derived Tumors

High-grade serous carcinoma (HGSC) is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs) are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOELOW) was developed and continuously passaged in culture to mimic cellular aging (MOEHIGH). The MOEHIGH cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOEHIGH cells proliferated significantly faster than MOELOW, and the MOEHIGH cells produced more 2D foci and 3D soft agar colonies as compared to MOELOW. MOEHIGH were xenografted into athymic female nude mice both in the subcutaneous and the intraperiteonal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOEHIGH and MOELOW were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOEHIGH had enhanced protein expression of c-myc, Cyclin E, p53 and FOXM1 with reduced expression of p21. MOEHIGH were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans

Signaling Pathways Contributing to High-Grade Serous Ovarian Cancer and Metastasis from Fallopian Tube

The fallopian tube epithelium (FTE) gives rise to high grade serous ovarian cancer (HGSOC), the most lethal gynecological malignancy. A mutation in p53 is reported in 96% of HGSOC. A transgenic mouse model and a cellular model with p53 mutation was developed and determined that p53 mutation alone is not sufficient to form tumors, however it significantly induced the cell migration. In vitro and In vivo analyses revealed that in FTE, mutant p53 reduced CDH6 (cell adhesion protein), through direct binding on CDH6 promoter, but CDH6 is not expressed in the ovaries. The existence of a FTE specific mutant p53 marker, may add to the existing tools for finding the cell of origin of HGSOC and may improve personalized therapies that work better in tumors arising from the FTE. In order to identify key drivers of HGSOC tumorigenesis, a spontaneous model of FTE derived cancer (MOEhigh - murine oviductal epithelium high passage, equivalent to human FTE) was made that contained gene alterations concordant to HGSOC. A human prolactin (PRL) like gene, Prl2c2 was amplified >100 fold in the model. Prl2c2 stable knockdown in MOEhigh cells demonstrated a significant reduction in cell proliferation, 2-dimensional foci, anchorage independent growth, and completely blocked tumor formation. The overall survival of ovarian cancer patients from transcriptome analysis of 1868 samples was lower when abundant PRL and prolactin receptors (PRL-R) were expressed. A HGSOC cell line (OVCAR3) and a tumorigenic human FTE cell line (FT33-Tag-Myc) were treated with recombinant PRL and a significant increase in cellular proliferation was detected. A CRISPR/Cas9 mediated PRL-R deletion in OVCAR3 and FT33-Tag-Myc cells demonstrated significant reduction in cell proliferation and eliminated tumor growth using the OVCAR3 model. PRL phosphorylated STAT5, m-TOR and ERK in ovarian cancer cells. Until now, PRL serum levels were only identified as a diagnostic marker for screening OVCA. However, this study for the first time, report that PRL is expressed and mediates tumorigenesis in human FTE. Small molecules were identified to block PRL-R activity and thereby providing a novel strategy, which with further clinical validation can be used to prevent HGSOC tumor formation from fallopian tube

A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation

Abstract Introduction Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality. Results Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole–cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23–79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85–99%. Conclusions Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole–cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield