Search

The marine pathogen Vibrio vulnificus senses and responds to environmental stimuli via two chemosensory systems and 42-53 chemoreceptors. Here, we present an analysis of the V. vulnificus Aer2 chemoreceptor, VvAer2, which is the first V. vulnificus chemoreceptor to be characterized. VvAer2 is related to the Aer2 receptors of other gammaproteobacteria, but uncharacteristically contains three PAS domains (PAS1-3), rather than one or two. Using an E. coli chemotaxis hijack assay, we determined that VvAer2, like other Aer2 receptors, senses and responds to O

Simple Summary Ovarian cancer has a dismal prognosis and innovative treatment options are necessary to improve survival. Because the microRNA let-7 is often lost in this and other cancers, and its loss is associated with poor prognosis, we focused on therapeutic strategies to replace it. We report that let-7 overexpression in patient-derived cells resulted in a loss of aggressiveness: inhibition of migration and invasion (associated with metastasis), repression of cancer stem cell attributes (necessary for tumor maintenance and recurrence), and promotion of cell death (required for sensitivity to chemotherapy drugs). Further, cells in which let-7 is overexpressed were more sensitive to PARP inhibitors, even in patients who otherwise could not benefit from these drugs. We show that let-7 reduces the expression of several genes that may contribute to these effects. These actions of let-7 add to the rationale for use of this miRNA as a treatment for selected ovarian cancer patients. Abstract High-grade serous carcinoma of the ovary is a deadly gynecological cancer with poor long-term survival. Dysregulation of microRNAs has been shown to contribute to the formation of cancer stem cells (CSCs), an important part of oncogenesis and tumor progression. The let-7 family of microRNAs has previously been shown to regulate stemness and has tumor suppressive actions in a variety of cancers, including ovarian. Here, we demonstrate tumor suppressor actions of let-7i: repression of cancer cell stemness, inhibition of migration and invasion, and promotion of apoptosis, features important for cancer progression, relapse, and metastasis. Let-7i over-expression results in increased sensitivity to the PARP inhibitor olaparib in samples without BRCA mutations, consistent with induction of BRCAness phenotype. We also show that let-7i inhibits the expression of several factors involved in the homologous recombination repair (HRR) pathway, providing potential mechanisms by which the BRCAness phenotype could be induced. These actions of let-7i add to the rationale for use of this miRNA as a treatment for ovarian cancer patients, including those without mutations in the HRR pathway.

We aimed to determine the mechanism of epithelial-mesenchymal transition (EMT)-induced stemness in cancer cells. Cancer relapse and metastasis are caused by rare stem-like cells within tumors. Studies of stem cell reprogramming have linked let-7 repression and acquisition of stemness with the EMT factor, SNAI1. The mechanisms for the loss of let-7 in cancer cells are incompletely understood. In four carcinoma cell lines from breast cancer, pancreatic cancer and ovarian cancer and in ovarian cancer patient-derived cells, we analyzed stem cell phenotype and tumor growth via mRNA, miRNA, and protein expression, spheroid formation, and growth in patient-derived xenografts. We show that treatment with EMT-promoting growth factors or SNAI1 overexpression increased stemness and reduced let-7 expression, while SNAI1 knockdown reduced stemness and restored let-7 expression. Rescue experiments demonstrate that the pro-stemness effects of SNAI1 are mediated via let-7. In vivo, nanoparticle-delivered siRNA successfully knocked down SNAI1 in orthotopic patient-derived xenografts, accompanied by reduced stemness and increased let-7 expression, and reduced tumor burden. Chromatin immunoprecipitation demonstrated that SNAI1 binds the promoters of various let-7 family members, and luciferase assays revealed that SNAI1 represses let-7 transcription. In conclusion, the SNAI1/let-7 axis is an important component of stemness pathways in cancer cells, and this study provides a rationale for future work examining this axis as a potential target for cancer stem cell-specific therapies.Novelty and ImpactThis study provides new insight into molecular mechanisms by which EMT transcription factor SNAI1 exerts its pro-stemness effects in cancer cells, demonstrating its potential as a stem cell-directed target for therapy. In vitro and in vivo, mesoporous silica nanoparticle-mediated SNAI1 knockdown resulted in restoration of let-7 miRNA, inhibiting stemness and reducing tumor burden. Our studies validate in vivo nanoparticle-delivered RNAi targeting the SNAI1/let-7 axis as a clinically relevant approach.

We studied ovarian cancer patient‐derived cells to determine their epithelial vs. mesenchymal phenotype, and their stemness, migration, invasion, and tumor growth characteristics. Surprisingly, stemness could be dissociated from invasiveness. We observed that lower let‐7 levels are associated with the epithelial state and stemness, reliably predict self‐renewal and tumor burden in mice, and could contribute to prognosis calculations., Patient‐derived samples present an advantage over current cell line models of high‐grade serous ovarian cancer (HGSOC) that are not always reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we set out to characterize a panel of patient‐derived cells and determine their epithelial and mesenchymal characteristics. We analyzed RNA and protein expression levels in patient‐derived xenograft (PDX) models of HGSOC, and functionally characterized these models using flow cytometry, wound healing assays, invasion assays, and spheroid cultures. Besides in vitro work, we also evaluated the growth characteristics of PDX in vivo (orthotopic PDX). We found that all samples had hybrid characteristics, covering a spectrum from an epithelial‐to‐mesenchymal state. Samples with a stronger epithelial phenotype were more active in self‐renewal assays and more tumorigenic in orthotopic xenograft models as compared to samples with a stronger mesenchymal phenotype, which were more migratory and invasive. Additionally, we observed an inverse association between microRNA let‐7 (lethal‐7) expression and stemness, consistent with the loss of let‐7 being an important component of the cancer stem cell phenotype. We observed that lower let‐7 levels were associated with the epithelial state and a lower epithelial mesenchymal transition (EMT) score, more efficient spheroid and tumor formation, and increased sensitivity to platinum‐based chemotherapy. Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: Cells with lower let‐7 levels were more tumorigenic, but less migratory, and with a lower EMT score, than those with higher let‐7 levels. We conclude that let‐7 expression and epithelial/mesenchymal state are valuable predictors of HGSOC proliferation, in vitro self‐renewal, and tumor burden in vivo.

Ovarian cancer ranks fifth among the causes of cancer death in US women. Despite initial positive response to cytoreductive surgery and chemotherapy treatments with platinum-based therapies, over 80% of patients with high-grade serous ovarian cancer (HGSOC), the most common type of ovarian cancer, exhibit relapse. Most recurrent cases of HGSOC are resistant to chemotherapy, and cells are highly invasive. Therefore, further studies are required to understand the mechanisms of invasiveness and chemoresistance in relapse patients. In HGSOC, invasiveness can result when epithelial cancer cells undergo epithelial-mesenchymal transition (EMT). The transcription factor SNAI1 (Snail) initiates the EMT process and increased Snail expression can result in enhancement of metastatic potential, acquisition of stem cell-like characteristics, and resistance to chemotherapeutic drugs. However, the mechanism of activation of invasiveness in ovarian cancer by Snail has not been defined. In OVCAR8, a HGSOC cell line, comparison of shSnail knockdown to control through RNA-sequencing revealed that Snail expression is directly correlated to matrix metalloprotease 1 (MMP1) expression. MMP1 is an extracellular matrix remodeler known to digest interstitial collagens. High MMP1 expression has been linked to increased cancer invasion, metastasis, angiogenesis, and apoptosis. Furthermore, in breast cancer, MMP1 has been reported to be a downstream target of Slug, another major EMT transcription factor. Some evidence for Snail activation of MMPs in liver cancer has been presented, but a direct relationship has not been established. Therefore, we hypothesize that overexpression of Snail in ovarian cancer cells results in MMP1 upregulation, leading to increased invasion. RT-qPCR was performed to analyze relative mRNA expression levels of Snail and MMP1 in eight cell lines. We found that Snail and MMP1 levels were higher in the more mesenchymal-like cell lines, some of which are known to be more invasive. Additionally, Snail overexpression led to a significant corresponding increase in MMP1 mRNA expression and resistance to cisplatin. Chromatin immunoprecipitation (ChIP) experiments demonstrate Snail binding to the MMP1 promoter. Thus, our preliminary findings have shown strong evidence of a Snail-mediated regulation of MMP1 expression in the context of HGSOC invasiveness. Citation Format: Tise Suzuki, Casey Curow, Hanmin Wang, Asis Martinez, Nozomi Hojo, Juli Unternaehrer. Snail-mediated regulation of MMP1 expression in the context of ovarian cancer invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2881.

Objective: Patient-derived samples present an advantage over current cell line models of high grade serous ovarian cancer (HGSOC), which are flawed in terms of being reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we characterized a panel of patient-derived cells to determine their epithelial and mesenchymal characteristics, invasiveness, proliferation, stemness, and in vivo growth. Experimental procedures: Patient-derived xenograft (PDX) models of HGSOC were analyzed in vitro for phenotypic (RNA, let-7 miRNA, and protein expression, flow cytometry) and functional aspects including growth in spheroids, wound healing assays, invasion assays and in vivo (orthotopic PDX) growth characteristics. Results: Samples fell along the spectrum from epithelial to mesenchymal, and all had hybrid characteristics. Those toward the more epithelial end of the spectrum were most active in self-renewal assays, and grew most robustly in orthotopic xenograft models. Chemoresistance correlated both with the mesenchymal state and with BRCA2 wild type status. Loss of microRNA let-7 (lethal-7) is an important component of the cancer stem cell phenotype, and we observed an inverse association between let-7 expression and the epithelial state. We observed lower levels of let-7, more efficient spheroid and tumor formation, and increased sensitivity to platinum-based chemotherapy in cells with the most epithelial phenotype. Conclusions: Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: epithelial cells (those with least let-7 expression) were less migratory, but more tumorigenic, than the mesenchymal cells with higher let-7 expression. We conclude that epithelial/mesenchymal state and let-7 expression are valuable predictors of HGSOC proliferation, in vitro self-renewal, and tumor burden in vivo. Citation Format: Juli Unternaehrer, Evgeny Chirshev, Nozomi Hojo, Antonella Bertucci, Linda Sanderman, Anthony Nguyen, Hanmin Wang, Tise Suzuki, Emmanuel Brito, Shannalee Martine, Christine Castañón, Saied Mirshahidi, Marcelo Vazquez, Kerby C. Oberg, Yevgeniya J. Ioffe. Epithelial-mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with let-7 levels and predicts tumor growth and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1682.

Introduction: Cell fate choices are made throughout development, and in adult organisms whenever stem cells divide. We study the interrelation of transcription factors (TF) and microRNAs in cell fate decisions. Metastasis of ovarian and other solid tumors involves epithelial-mesenchymal transition (EMT), brought about by TF such as Snail, Twist, Slug, and Zeb1. Cells undergoing reprogramming paradoxically upregulate the EMT TF Snail and downregulate let-7, and Snail binds the promoters of several let-7 family members consistent with direct regulation. Let-7 is a microRNA highly expressed in differentiated cells, and absent in pluripotent cells. Its targets include the oncogenes Myc, Ras, and HMGA2. Many aspects of somatic cell reprogramming are seen as analogous to the acquisition of stem cell phenotypes observed in cancer. Let-7 levels are decreased in many ovarian tumors, and low let-7 correlates with poor prognosis. In breast cancer, let-7 downregulation is associated with metastasis. Results: Let-7 levels are known to be regulated by control of processing by the RNA binding protein and pluripotency factor Lin28, but transcriptional regulators of let-7 have not been described. We show that Snail represses let-7 transcription in cancer-related EMTs. ChIP assays demonstrate binding of Snail to let-7 promoters, and luciferase assays demonstrate direct regulation of Let-7i by Snail. Thus we are exploring two avenues for metastasis inhibition in OC: decreasing Snail and increasing let-7. We have characterized several OC cell lines for their phenotype with regard to EMT and cancer stem cell markers, in order to select agents to advance to testing in our orthotopic patient-derived xenograft model. We have examined the role of Snail in OC metastasis by manipulating expression levels and carrying out invasiveness assays. In epithelial and cancer cell lines, stimulation with TGF-beta or EGF caused morphological and gene expression changes consistent with EMT. The OC line OVSAHO underwent EMT when stimulated with EGF, but not with TGF-b. In breast, pancreatic, and OC cell lines, increases in Snail expression correlate with decreased let-7 expression. Conclusions: We propose that the inverse relationship between Snail and let-7 plays an important role at decision points crucial for cell fate decisions. In cancer metastasis, derepression of let-7 targets (many of them oncofetal genes) leads to dedifferentiation and a more aggressive phenotype. In addition to its role in directly causing EMT and affording cells with the migratory and invasive phenotype necessary for metastasis, our model places Snail at an important control point for initiation of dedifferentiation events normally held in check by let-7. Citation Format: Yevgeniya Ioffe, Alyse Hill, Michael McCarthy, Linda Sanderman, Hugo Campos, Tise Suzuki, Andrew Caster, Juli Unternaehrer-Hamm. The epithelial-mesenchymal transition factor Snail represses the tumor suppressor microRNA let-7 and contributes to invasiveness of ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B52.