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Supplementary Figure S1. Survivin and EMT markers were stained in sections of ovarian tumor of survivin KO and control mice; Supplementary Figure S2. Ovarian cancer cell migration and invasion following inhibition of apoptosis; Supplementary Figure S3. Cell viability in the upper chamber following treatment of MX106 in the migration assay; Supplementary Figure S4. Cell viability in the upper chamber following treatment of MX106 in the invasion assay; Supplementary Figure S5. SMAD dependent reporter gene luciferase activity; Supplementary Figure S6. TGFβ did not activate non-SMAD pathway in ovarian cancer cells; Supplementary Figure S7. Immunostaining for survivin and EMT markers in sections of ovarian tumor tissue following MX106 or vehicle treatment.

Survivin, a member of the inhibitor of apoptosis family, is upregulated in multiple cancers including ovarian cancer, but is rarely detectable in normal tissues. We previously reported that survivin promoted epithelial-to-mesenchymal transition (EMT) in ovarian cancer cells, suggesting that survivin may contribute to ovarian tumor metastasis and chemoresistance. In this study, we tested whether knockout or pharmacologic inhibition of survivin overcomes chemoresistance and suppresses tumor metastasis. The genetic loss of survivin suppressed tumor metastasis in an orthotopic ovarian cancer mouse model. To pharmacologically test the role of survivin on ovarian tumor metastasis, we treated chemo-resistant ovarian cancer cells with a selective survivin inhibitor, MX106, and found that MX106 effectively overcame chemoresistance in vitro. MX106 inhibited cell migration and invasion by attenuating the TGFβ pathway and inhibiting EMT in ovarian cancer cells. To evaluate the efficacy of MX106 in inhibiting ovarian tumor metastasis, we treated an orthotopic ovarian cancer mouse model with MX106, and found that MX106 efficiently inhibited primary tumor growth in ovaries and metastasis in multiple peritoneal organs as compared with vehicle-treated control mice. Our data demonstrate that inhibition of survivin using either genetic knockout or a novel inhibitor MX106 suppresses primary ovarian tumor growth and metastasis, supporting that targeting survivin could be an effective therapeutic approach in ovarian cancer.

Supplementary Figure 5. ATX and LPA receptor profiling in isolated rat alveolar type I-like (A) or type II (B) epithelial cells. Note that expression levels in type II alveolar cells are approximately 10-fold lower than in type I-like cells. Data presented are representative of an experiment performed three times in quadruplicates and are expressed as mean {plus minus} SD.

Supplementary Figure 3. (A) ATX and LPA receptor profiling in B16F10 cells transduced with GFP lentivirus using quantitative real-time PCR. Data are expressed as mean {plus minus} SD of an experiment performed twice in quadruplicates. (B) Representative images of lungs harvested at day 21 from mice that were injected with PBS (left panel, control) and mice that were injected with 1 x 105 GFP-expressing B16F10 cells (right panel). Arrow denotes a single fluorescent tumor nodule on lung surface. Scale bar = 200 microns.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein–coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors.Implications: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis. Mol Cancer Res; 13(1); 174–85. ©2014 AACR.

Supplementary Figure 2. LPA1-5 receptor profiling in whole lung tissue isolated from WT, LPA1-, LPA2- and LPA5KO mice, respectively using quantitative real-time PCR. Data are expressed as mean {plus minus} SEM (n = 3 mice).

Supplementary Figure 1. Time course of ATX activity measured in the conditioned media (CM) of (A) MM1 rat hepatocarcinoma, (B) HUVEC, (C) RLMVEC, and (D) isolated murine mesothelial cells. Control represents media that were not exposed to cells. Data are expressed as mean {plus minus} SD of an experiment performed three times in triplicates.

Supplementary Figure 4. Effect of BMP22 on the basal invasion of B16F10 cells across a matrigel layer. Data are representative of an experiment performed twice in quadruplicates and are expressed as mean {plus minus} SD. *Denotes p value < 0.05 using one-way ANOVA followed by a Bonferroni post-test.

Supplementary Table 1. Primer sequences used in quantitative Real-Time PCR analysis.

Supplementary Figures 1-5 from IFN Induces miR-21 through a Signal Transducer and Activator of Transcription 3–Dependent Pathway as a Suppressive Negative Feedback on IFN-Induced Apoptosis

Survivin, a member of the inhibitor of apoptosis family, is upregulated in multiple cancers including ovarian cancer, but is rarely detectable in normal tissues. We previously reported that survivin promoted epithelial-to-mesenchymal transition (EMT) in ovarian cancer cells, suggesting that survivin may contribute to ovarian tumor metastasis and chemoresistance. In this study, we tested whether knockout or pharmacologic inhibition of survivin overcomes chemoresistance and suppresses tumor metastasis. The genetic loss of survivin suppressed tumor metastasis in an orthotopic ovarian cancer mouse model. To pharmacologically test the role of survivin on ovarian tumor metastasis, we treated chemo-resistant ovarian cancer cells with a selective survivin inhibitor, MX106, and found that MX106 effectively overcame chemoresistance in vitro. MX106 inhibited cell migration and invasion by attenuating the TGFβ pathway and inhibiting EMT in ovarian cancer cells. To evaluate the efficacy of MX106 in inhibiting ovarian tumor metastasis, we treated an orthotopic ovarian cancer mouse model with MX106, and found that MX106 efficiently inhibited primary tumor growth in ovaries and metastasis in multiple peritoneal organs as compared with vehicle-treated control mice. Our data demonstrate that inhibition of survivin using either genetic knockout or a novel inhibitor MX106 suppresses primary ovarian tumor growth and metastasis, supporting that targeting survivin could be an effective therapeutic approach in ovarian cancer.

Introduction: Survivin, a member of the inhibitor of apoptosis family, is upregulated in multiple cancers including ovarian cancer, but is rarely detectable in normal tissues. Therefore, it is a potential drug target. However, targeting survivin is still challenge and small molecule inhibitors failed in clinical trials. It's very important to understand how survivin contributes to tumor development. In this study, we investigated the role of survivin in ovarian cancer using ovarian cancer cell lines in vitro and mouse models in vivo and examined how survivin contributes to ovarian primary ovarian tumor growth and metastasis by examining epithelial to mesenchymal transition (EMT). Methods: To define the role of survivin in EMT in ovarian cancer cells, we established survivin knockout cell lines using lentiviral CRISPR/Cas9 nickase and pharmacologically inhibited survivin with a novel inhibitor MX106. We examined EMT markers, migration and invasion assays in survivin disrupted ovarian cancer cells using Transwell plates. We also assessed primary ovarian tumor growth and metastasis in an orthotopic ovarian cancer mouse models through intrabursal injecting survivin KO cell line and also treatment using MX106. Findings: We found that disruption of survivin expression inhibited EMT by attenuating the TGFβ pathway in ovarian cancer cells. Cell proliferation, migration, and invasion were significantly inhibited in survivin knockout or MX106 treatment in SKOV3 and OVCAR3 cell lines compared to control cells. Inhibition of survivin sensitized cell responses to paclitaxel treatment and overcame chemoresistance in vitro. Lentiviral CRISPR/Cas9 mediated survivin knockout or MX106 treatment efficiently inhibited primary ovarian tumor growth and metastasis in orthotopic ovarian cancer mouse models. Conclusions: Our data demonstrate that inhibition of survivin using either genetic knockout or a novel inhibitor MX106 suppresses primary ovarian tumor growth and metastasis by attenuating TGFβ pathway. MX106, as a novel survivin inhibitor effectively overcame chemoresistance compared to commercial YM155, which provides a novel approach for ovarian cancer therapy. Citation Format: Junming Yue. Genetic and pharmacological disruption of survivin suppressed primary and metastatic tumor growth by attenuating TGFB pathway in ovarian cancer [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 4184.

Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein–coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors. Implications: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis. Mol Cancer Res; 13(1); 174–85. ©2014 AACR.

Due to peritoneal metastasis and frequent recurrence, ovarian cancer has the highest mortality among gynecological cancers. Epithelial to mesenchymal transition (EMT) contributes to ovarian tumor metastasis. In this study, we report for the first time that metal regulatory transcription factor 1 (MTF1) was upregulated in ovarian cancer, and its high expression was associated with poor patient survival and disease relapse. Knockout of MTF1 using lentiviral CRISPR/Cas9 nickase vector-mediated gene editing inhibited EMT by upregulating epithelial cell markers E-cadherin and cytokeratin 7, and downregulating mesenchymal markers Snai2 and β-catenin in ovarian cancer SKOV3 and OVCAR3 cells. Loss of MTF1 reduced cell proliferation, migration, and invasion in both SKOV3 and OVCAR3 cells. Knockout of MTF1 upregulated the expression of the KLF4 transcription factor, and attenuated two cellular survival pathways, ERK1/2 and AKT. Our studies demonstrated that MTF1 plays an oncogenic role and contributes to ovarian tumor metastasis by promoting EMT. MTF1 may be a novel biomarker for early diagnosis as well as a drug target for clinical therapy. Citation Format: Guannan Zhao, Lawrence M Pfeffer Junming Yue. KNOCKOUT OF MTF1 RESULTS IN THE INHIBITION OF EMT IN OVARIAN CANCER CELLS [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 NT-120.

The microRNA miR-21 is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. Here, we report that the cytokine IFN rapidly induces miR-21 expression in human and mouse cells. Signal transducer and activator of transcription 3 (STAT3) was implicated in this pathway based on the lack of IFN effect on miR-21 expression in prostate cancer cells with a deletion in the STAT3 gene. STAT3 ablation abrogated IFN induction of miR-21, confirming the important role of STAT3 in regulating miR-21. Chromatin immunoprecipitation analysis showed that STAT3 directly bound the miR-21 promoter in response to IFN. Experiments in mouse embryo fibroblasts with a genetic deletion of the p65 NF-κB subunit showed that IFN-induced miR-21 expression was also dependent on NF-κB. STAT3 silencing blocked both IFN-induced p65 binding to the miR-21 promoter and p65 nuclear translocation. Thus, IFN-induced miR-21 expression is coregulated by STAT3 and NF-κB at the level of the miR-21 promoter. Several cell death regulators were identified as downstream targets of miR-21, including PTEN and Akt. Functional experiments in prostate cancer cells directly showed that miR-21 plays a critical role in suppressing IFN-induced apoptosis. Our results identify miR-21 as a novel IFN target gene that functions as a key feedback regulator of IFN-induced apoptosis. Cancer Res; 70(20); 8108–16. ©2010 AACR.

Autotaxin (ATX), a lysophospholipase D that generates the growth factor-like mediator lysophosphatidic acid LPA, is up-regulated in metastatic and chemotherapy-resistant carcinomas. Many cancer cells secrete ATX, and it contributes to their invasive properties. The biological functions of ATX are mediated by LPA acting on G protein-coupled receptors (LPAR1-8). Many cancers overexpress multiple subtypes of LPAR and the overexpression of LPAR leads to malignant transformation, metastasis, and resistance to therapy. We have previously shown that knockdown of ATX expression in B16F10 melanoma cells decreased tumor invasion in vitro and lung metastasis in vivo. In our present study, we sought to understand the roles of LPAR both in the tumor cells and stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express the LPA5 receptor. We found that these cells do not invade across a matrigel layer in response to LPA. shRNA knockdown of LPA5 receptors in B16F10 attenuates this inhibition, suggesting that the LPA5 receptor acts as an anti-invasive receptor in these tumor cells. Interestingly, we identified a different role for host LPA5 receptor. We found that the incidence of B16F10 lung metastasis was significantly reduced in the LPA5 knockout mice compared to wild type mice. In addition, LPA1 knockout mice also showed diminished B16F10 lung metastasis suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of these knockout animals was apparent at 24 hours after injection. However, deletion of LPA1 or LPA5 did not affect the subcutaneous growth of B16F10 tumors. In summary, our results suggest that LPAR, in particular LPA5 and LPA1 receptors in tumor and stromal cells may play different roles in invasion and the seeding of metastasis. Citation Format: Sue-Chin Lee, Yuko Fujiwara, Jianxiong Liu, Junming Yue, Ryoko Tsukahara, Erzsebet Szabo, Renukadevi Patil, Duane D. Miller, Louisa Balazs, Tamas Oravecz, Gabor J. Tigyi. Autotaxin, LPA1 and LPA5 receptors in the tumor microenvironment determine melanoma invasion and metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B28. doi:10.1158/1538-7445.CHTME14-B28