Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

Endometriosis is a chronic, estrogen-dependent inflammatory condition that is marked by the growth of lesions outside the uterus, along with angiogenesis, fibrosis, immune dysregulation, and pain sensitization. There is now growing evidence that receptor tyrosine kinase (RTK) signaling networks are central to these processes, integrating proliferative, angiogenic, inflammatory, endocrine, and microenvironmental signals. This review summarizes the roles of six major RTK axes in endometriosis and evaluates their receptor-specific mechanisms, therapeutic relevance, and modulation by representative phytochemicals. An English-language literature search of PubMed was conducted with no publication date restrictions using combinations and variations of the following keywords: phytochemicals, endometriosis, EGFR, VEGFR, IGF1R, PDGFR, FGFR, MET, curcumin, resveratrol, epigallocatechin gallate (EGCG), quercetin, naringenin, berberine, apigenin, luteolin, genistein, and baicalein. Additional search terms related to receptor signaling mechanisms, downstream pathways, cellular phenotypes, and therapeutic relevance were also incorporated to ensure comprehensive coverage of the literature. The RTK signaling pathways control stromal activation, lesion survival, angiogenic support, extracellular matrix remodeling, neuroimmune sensitization, and malignant progression. Of the six RTK axes examined, EGFR, VEGFR, and IGF1R have the strongest mechanistic and pharmacological support, whereas PDGFR, FGFR, and MET remain biologically plausible targets. Even though phytochemicals do not consistently act as receptor-selective RTK inhibitors, evidence shows they reduce RTK-centred signaling networks by targeting common downstream hubs. As a result of their effects at the network level, the phytochemicals may help to suppress lesion proliferation, angiogenesis, invasion, fibrosis, inflammation, oxidative stress, and pain sensitization. Endometriosis should be viewed as an RTK-centered network disease rather than a single-pathway disorder. In this context, phytochemicals act as modulators that attenuate signaling hubs, offering a mechanistically aligned strategy for a disease driven by redundancy and microenvironmental crosstalk.

Purpose: Ovulation is a strong risk factor for high grade serous carcinoma (HGSC) of ovary known to largely originate from the fallopian tube fimbriae. Previously, we have showed ROS in the mature follicular fluid (FF) is mutagenic and cytotoxic to fimbria epithelial cells, and induces DNA double strand breaks (DSB) in the context of p53 loss. We hypothesized that hemoglobin (Hb) in FF accumulated in cul-de-sac (CDS) after ovulatory bleeding may protect the bathed fimbria from cell death and enhance cancer initiation.

Ovulation is the strongest risk factor for ovarian high-grade serous carcinoma (HGSC) that largely originates from the fallopian tube fimbriae and always carries loss-of-function mutations of TP53 in both early and late lesions. Mature ovarian follicle contains high level of reactive oxygen species (ROS). When released from ovulation, follicular fluid (FF) bathes the fimbriae and may lead to DNA double strand break (DSB) and neoplastic transformation. In this study, we examined the mutagenic and tumorigenic activities of human pre-ovulatory FFs. A subset (6/11) of FFs was found with high levels of ROS whereas the antioxidant capacities were indifferent. These ROS-high FFs induced intracellular ROS and DSBs in the secretory cell population of fimbriae epithelium. In the context of p53 and Rb deficiency in an E6/E7 immortalized fimbria secretory cell line, FF-exposed secretory cells overcame apoptosis and maintained the population carrying ROS and DSB. The cancer initiation effects of FF were further recapitulated in Trp53^-/- mice. When introduced into the mammary fat pad, ROS-high but not ROS-low FFs induced early-onset B-cell lymphoma. Cotreatment with physiological concentration of melatonin, a potent antioxidant, ameliorated the mutagenic and tumorigenic effect of ROS-high FF in vitro and in vivo. The study revealed ROS and mitogens in mature ovarian follicles could initiate the transformation of fimbria epithelium in the context of p53 loss and melatonin is a potent preventive agent.

High grade serous carcinoma (HGSC) of the ovary constitutes the majority of incidence and death of ovarian cancer, mainly because of the elusive etiology and cell of origin. Ovulation has been regarded as a strong risk factor and use of oral contraceptives (OC) as a protective factor for ovarian cancer with obvious dose (the span of ovulation life and duration of OC use) effect, but the mechanism is unknown. Meanwhile, more and more evidences have indicated the fallopian tube fimbriae to be the tissue of origin of HGSC. We hypothesize that high level of reactive oxygen species (ROS), which is indispensible for ovulation, as well as abundant mitogens in the mature follicular fluid is responsible for the initiation (DNA double strand breaks, DSB) and promotion (proliferation) of carcinogenesis to the fallopian tube secretory cells. We first tested the scrapped cells of the human fallopian tube epithelium for the extending of DSB. By flow cytometry with gating for the secretory cell marker PAX8 and DSB marker γH2AX, we found the secretory cells in the fimbriae harbored significantly more DSB than the those in the proximal tube. In an immortalized cell line of the human fimbriae secretory cell (we named FE25), we tested the mutagenic and mitogenic effects of the ROS and antioxidant in human follicular fluids at ovulation (FF). We found a balanced activity of of ROS and antioxidants in the undiluted follicular fluid with the antioxidants to be limited, such that, upon serial dilution, the intracellular ROS level raised proportionally. This dilution-raised effect of ROS was able to induce γH2Ax accumulation in the FE25 cells. Upon treating with sub-nM level of melatonin, the most potent and major antioxidant in FF, the level of intracellular ROS and γH2Ax readily decreased. Temporally, we found a two-stage effect of FF on the survival and proliferation of FE25, with an early phase of cell death, which occurred within 12 hours after FF treatment, followed by a cell expansion phase occurring at day 2. We interpret this two-stage effect to be an immediate ROS-induced DSB and cell apoptosis followed by a mitogen-induced proliferation of the survived cells. These survived cells carried the DSB and may be clonally expanded and move on to the carcinogenic road. We conclude that an imbalance favoring the ROS activity in the FF, likely from a significant dilution of FF by an unknown pathological status (such as fimbriae inflammation or ascites), may induce an oxidative stress and DSB in the fallopian tube fimbriae. Cells that survive this stress with some carrying DSB are expanded by the rich mitogens in the same FF. When enough mutations of critical genes were accumulated in a stem cell in this secretory population, cancer lesion such as STIC can thus arise.

Recent studies have unveiled the fallopian tube fimbria as the major tissue-of-origin and incessant ovulation as the main etiological risk factor of ovarian high-grade serous carcinoma (HGSC) but the mechanism of cancer initiation remains elusive. In a series of study we have found a subset of preovulatory follicular fluids (FF) contain high level of ROS that induce DNA double strand breaks as well as apoptosis to the fimbrial epithelial cells in vitro and in vivo. We also found hemoglobin in pelvis, most likely contributed by retrograde menstruation of previous cycles, reduces the ovulatory ROS level extracellularly and results in a shifting from the apoptotic to the survival fate of ROS-stressed fimbrial epithelial cells. These initiating factors in FF, although may contributes to the TP53 mutation and other early genetic hits in the genesis of HGSC, cannot fully transform the immortalized fimbrial epithelial cells, with loss of p53 and Rb. In this study, we demonstrated that IGF axis proteins, including IGFBP2- and IGFBP6-bound IGF2 as well as the IGFBP-lytic enzyme PAPP-A are abundantly present in ovulatory FF, and disclosed their effect on the fallopian tube fimbrial epithelium. Upon engaging with glycosaminoglycans (GAG) on the membrane of fimbrial epithelial cells, PAPP-A cleaves IGFBPs and releases bioactive IGF2 in close proximity to its receptor IGF-1R. This leads to stemness activation and clonal expansion of primary and immortalized fimbrial epithelial cells, and to malignant transformation of p53/Rb- and p53/CCNE1-disrupted fimbrial secretory cells. By antibody depletion, we proved each component of this IGF axis is essential and confers the majority of the transformation/regeneration activities in FF. Two signaling pathways, IGF-1R/ AKT/mTOR and IGF-1R/AKT/NANOG, are responsible for the phenotypes. Among them, IGF-1R and NANOG were critical since blocking their expression or function lead to a complete loss of the transformation. Given that ovulation is an acute inflammatory process inevitably damaging the ovarian surface and the fimbriae, IGF axis in FF takes a role of tissue repair and, in situations when the ROS-induced TP53/Rb mutations accumulated in stem cells, may lead to carcinogenesis of the fallopian tube fimbria. Citation Format: Tang-Yuan Chu, Che-Fang Hsu, Hsuan-Shun Huang. IGF-AXIS SIGNALING DURING OVULATION IS RESPONSIBLE FOR THE STEMNESS CELL EXPANSION AND TRANSFORMATION OF FALLOPIAN TUBE FIMBRIAL EPITHELIUM [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-056.