Abnormal regulation and expression of microRNAs (miRNAs) has been documented in various diseases including cancer. The miRNA let - 7 (MIRLET7) family controls developmental timing and differentiation. Let - 7 loss contributes to carcinogenesis via an increase in its target oncogenes and stemness factors. Let - 7 targets include genes regulating the cell cycle, cell signaling, and maintenance of differentiation. It is categorized as a tumor suppressor because it reduces cancer aggressiveness, chemoresistance, and radioresistance. However, in rare situations let - 7 acts as an oncogene, increasing cancer migration, invasion, chemoresistance, and expression of genes associated with progression and metastasis. Here, we review let - 7 function as tumor suppressor and oncogene, considering let - 7 as a potential diagnostic and prognostic marker, and a therapeutic target for cancer treatment. We explain the complex regulation and function of different let - 7 family members, pointing to abnormal processes involved in carcinogenesis. Let - 7 is a promising option to complement conventional cancer therapy, but requires a tumor specific delivery method to avoid toxicity. While let - 7 therapy is not yet established, we make the case that assessing its tumor presence is crucial when choosing therapy. Clinical data demonstrate that let - 7 can be used as a biomarker for rational precision medicine decisions, resulting in improved patient survival.

To develop effective therapies for advanced high grade serous ovarian cancer (HGSOC), understanding mechanisms of recurrence and metastasis is necessary. In this study, we define the epithelial/mesenchymal status of cell lines that accurately model HGSOC, and evaluate the therapeutic potential of targeting Snai1 (Snail), a master regulator of the epithelial/mesenchymal transition (EMT) in vitro and in vivo. The ratio of Snail to E-cadherin (S/E index) at RNA and protein levels was correlated with mesenchymal morphology in four cell lines. The cell lines with high S/E index (OVCAR8 and COV318) showed more CSC-like, motile, and chemoresistant phenotypes than those with low S/E index (OVSAHO and Kuramochi). We tested the role of Snail in regulation of malignant phenotypes including stemness, cell motility, and chemotherapy resistance: shRNA-mediated knockdown of Snail reversed these malignant phenotypes. Interestingly, the expression of let-7 tumour suppressor miRNA was upregulated in Snail knockdown cells. Furthermore, knockdown of Snail decreased tumour burden in an orthotopic xenograft mouse model. We conclude that Snail is important in controlling HGSOC malignant phenotypes and suggest that the Snail/Let-7 axis may be an attractive target for HGSOC treatment.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 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.

Patients with metastatic castration-resistant prostate cancer (mCRPC) develop resistance to conventional therapies including docetaxel (DTX). Identifying molecular pathways underlying DTX resistance is critical for developing novel combinatorial therapies to prevent or reverse this resistance. To identify transcriptomic signatures associated with acquisition of chemoresistance we profiled gene expression in DTX-sensitive and -resistant mCRPC cells using RNA sequencing (RNA-seq). PC3 and DU145 cells were selected for DTX resistance and this phenotype was validated by immunoblotting using DTX resistance markers (e.g. clusterin, ABCB1/P-gp, and LEDGF/p75). Overlapping genes differentially regulated in the DTX-sensitive and -resistant cells were ranked by Gene Set Enrichment Analysis (GSEA) and validated to correlate transcript with protein expression. GSEA revealed that genes associated with cancer stem cells (CSC) (e.g., NES, TSPAN8, DPPP, DNAJC12, and MYC) were highly ranked and comprised 70% of the top 25 genes differentially upregulated in the DTX-resistant cells. Established markers of epithelial-to-mesenchymal transition (EMT) and CSCs were used to evaluate the stemness of adherent DTX-resistant cells (2D cultures) and tumorspheres (3D cultures). Increased formation and frequency of cells expressing CSC markers were detected in DTX-resistant cells. DU145-DR cells showed a 2-fold increase in tumorsphere formation and increased DTX resistance compared to DU145-DR 2D cultures. These results demonstrate the induction of a transcriptomic program associated with stemness in mCRPC cells selected for DTX resistance, and strengthen the emerging body of evidence implicating CSCs in this process. In addition, they provide additional candidate genes and molecular pathways for potential therapeutic targeting to overcome DTX resistance.

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.

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.

Cytokine receptor-like factor 2 B-cell acute lymphoblastic leukemia (CRLF2 B-ALL) is a high-risk subtype characterized by CRLF2 overexpression with poor survival rates in children and adults. CRLF2 and interleukin-7 receptor alpha (IL-7Rα) form a receptor for the cytokine thymic stromal lymphopoietin (TSLP), which induces JAK/STAT and PI3K/AKT/mTOR pathway signals. Previous studies from our group showed that low TSLP doses increased STAT5, AKT, and S6 phosphorylation and contributed to CRLF2 B-ALL cell survival. Here we investigated the role of TSLP in the survival and proliferation of CRLF2 B-ALL cells in vitro and in vivo. We hypothesized that high doses of TSLP increase CRLF2 signals and contribute to increased proliferation of CRLF2 B-ALL cells in vitro and in vivo. Interestingly, we observed the opposite effect. Specifically, high doses of TSLP induced apoptosis in human CRLF2 B-ALL cell lines in vitro, prevented engraftment of CRLF2 B-ALL cells, and prolonged the survival of +TSLP patient-derived-xenograft mice. Mechanistically, we showed that high doses of TSLP induced loss of its receptor and loss of CRLF2 signals in vitro. These results suggest that high doses of TSLP could be further investigated as a potential therapy for the treatment of CRLF2 B-ALL.

Purpose: We aimed to understand how stemness is regulated by the Snail/let-7 axis in ovarian cancer cells, toward the goal of developing novel treatments that target stem-like cells to sensitize chemoresistant tumors. Background: Metastatic progression and disease recurrence in high-grade serous ovarian carcinoma (HGSOC) are in part driven by epithelial-mesenchymal transition (EMT) and cell reversion to cancer stem cell (CSC) phenotype. A contributor to the CSC phenotype is loss of miRNA let-7, due to increased expression of its targets, including oncogenes and pluripotency factors. Mechanisms leading to loss of let-7 are incompletely understood. Methods: Snail was overexpressed by estrogen receptor fusion, or EMT was induced with epidermal growth factor (EGF). Snail knockdown (KD) was by lentiviral small hairpin (sh)RNA expression and puromycin selection. Cell surface expression of CSC markers was analyzed by flow cytometry. Expression at the protein level was detected by immunofluorescence microscopy and Western blot. Gene expression of mesenchymal, epithelial, and pluripotency factors and miRNAs was detected by q-RT-PCR. Migration was determined by scratch assay and live-cell imaging. Data were analyzed by linear regression. Chromatin immunoprecipitation of Snail was followed by qPCR. Let-7i promoter luciferase with and without Snail were co-transfected into 293T cell lines and followed by luciferase assays for detection of bioluminescence. Let-7 overexpression was by transfection of mimics. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six-week-old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized HGSOC cells (OVCAR8 vs PDX) with control vs. Snail KD (250-500K cells). Bioluminescence was quantified by IVIS Lumina III over 21-50 days and analyzed by Living Image software. Results: We characterized several cell lines that accurately model HGSOC for their epithelial (E) vs. mesenchymal (M) and stem cell properties. OVSAHO, COV318, and OVCAR8 were selected for further study in increasing order of mesenchymal characteristics. Increasing let-7 levels or decreasing Snail levels disrupted the stem cell phenotype, reduced cells’ migratory ability, and sensitized cells to cisplatin. Snail bound promotors of let-7 in cell lines tested. Luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic tumor burden was significantly reduced in orthotopic xenografts using OVCAR8 cells expressing Snail shRNA. Similarly, in PDX derived from ascites or ovarian tumors, there was reduced tumor burden in Snail knockdown animals. Conclusions: We present preliminary data showing that the EMT factor Snail represses let-7, placing Snail at the nexus of dedifferentiation via loss of let-7, and invasiveness via EMT. PDX reproducibly phenocopy HGSOC. Cell line and patient-derived data support the relationship between Snail expression, let-7 downregulation, and the induction of CSC. We hypothesize that Snail KD disrupts the cancer stem cell state and disrupts disease progression. We propose that Snail is a potential pharmaceutical target for recurrent, metastatic EOC. Citation Format: Alyse Huisken, Nozomi Hojo, Hanmin Wang, Evgeny Chirshev, Carlotta Glackin, Yevgeniya Ioffe, Julia J. Unternaehrer-Hamm. Mechanism of tumor suppressor miRNA let-7 downregulation in ovarian cancer: The epithelial-mesenchymal transition factor Snail is associated with stemness and represses let-7. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A73.