Inflammation can act as a crucial mediator of epithelial-to-mesenchymal transition (EMT). In this study, we show that oncostatin M (OSM) is expressed in an autocrine/paracrine fashion in invasive breast carcinoma. OSM stimulation promotes spontaneous lung metastasis of MCF-7 xenografts in nude mice. A conspicuous epigenetic transition was induced by OSM stimulation not only in breast cancer cell lines but also in MCF-7 xenografts in nude mice. The expression of miR-200 and let-7 family members in response to OSM stimulation was downregulated in a signal transducer and activator of transcription factor 3 (Stat3)-dependent manner, resulting in comprehensive alterations of the transcription factors and oncoproteins targeted by these microRNAs. Inhibition of Stat3 activation or the ectopic expression of let-7 and miR-200 effectively reversed the mesenchymal phenotype of breast cancer cells. Stat3 promotes the transcription of Lin-28 by directly binding to the Lin-28 promoter, resulting in the repression of let-7 expression and concomitant upregulation of the let-7 target, high-mobility group A protein 2 (HMGA2). Knock down of HMGA2 significantly impairs OSM-driven EMT. Our data indicate that downregulation of let-7 and miR-200 levels initiates and maintains OSM-induced EMT phenotypes, and HMGA2 acts as a master switch of OSM-induced EMT. These findings highlight the importance of Stat3-coordinated Lin-28B-let-7-HMGA2 and miR-200-ZEB1 circuits in the cytokine-mediated phenotypic reprogramming of breast cancer cells.
The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.
Tumor microenvironment has a crucial role in cancer development and progression, whereas the mechanism of how it regulates angiogenesis is unclear. In this study, we simulated the colorectal carcinoma microenvironment by conditioned medium (CM) of colorectal carcinoma cell lines (SW620, HT-29, HCT116) supernatant or colorectal carcinoma tissue homogenate supernatant to induce normal endothelial cells (NECs). We found that colorectal carcinoma CM promoted tumor angiogenesis by coercing NECs toward tumor endothelial cells (TECs) with the activation of the JAK/STAT3 signaling pathway. Antibody array analysis showed HT-29 supernatant contained numerous angiogenesis-related proteins, especially IL-8. Interestingly, the production of IL-8 in NECs induced by HT-29 CM was also increased. We also verified the crucial role of IL-8 in promoting the CM-induced angiogenesis, as IL-8 repression by neutralizing antibody abolished the transition of NECs toward TECs. Curcumin and (−)-epigallocatechin-3-gallate (EGCG) are broadly investigated in cancer chemoprevention. However, poor bioavailability hurdles their application alone, and the mechanism of their anti-angiogenesis still need to be illuminated. Here, we found that curcumin combination with EGCG attenuated the tumor CM-induced transition of NECs toward TECs by inhibiting JAK/STAT3 signaling pathway. Furthermore, the combination of curcumin and EGCG markedly reduced tumor growth and angiogenesis in the colorectal carcinoma PDX mouse model, and the combined anti-angiogenic effect was better than that of curcumin or EGCG alone. Taken together, our findings provide a new mechanism of tumor angiogenesis, and the combination of curcumin and EGCG represents a potential anti-angiogenic therapeutic method for colorectal carcinoma.
Inactivating germline mutations in the NF1 gene (encoding neurofibromin) cause neurofibromatosis type 1. In addition to peripheral nervous system tumors, NF1 patients are at higher risk for other cancers, including breast cancer. Tumor exome-sequencing studies demonstrate that approximately 20% of all human cancers have somatic NF1 mutations. NF1 has been best known for its ability to inactivate Ras as a GAP (GTPase Activating Protein). However, this function is served by a small GAP domain in a very large protein. Recurrent missense mutations inactivating the GAP activity are infrequent. In contrast, it is common to detect frameshift (FS) and nonsense (NS) NF1 mutations, which can create an NF1-null state deleting not only GAP, but also, potentially, undefined NF1 functions whose loss could also drive tumorigenesis. As we reported at SABCS previously, in 600+ patients treated by tamoxifen adjuvant monotherapy, we found that FS/NS NF1 mutations independently correlate with relapse risk (HR=2.6, p=0.03). To explore this finding, we silenced NF1 in preclinical models of ER+ breast cancer, which markedly enhanced ER transcriptional activities, causing estradiol (E2) hypersensitivity and converted tamoxifen into an agonist (in vitro and in vivo). Most important, these activities depend on ER, but not on NF1's GAP activity. These findings readily explain the poor patient outcomes associated with NS/FS NF1 mutations, and reveal a previously unrecognized function for NF1 in ER regulation. In the presence of an agonist, liganded ER repels co-repressors and recruits co-activators, while the reverse is true with an antagonist such as tamoxifen. Many co-regulators contain leucine/isoleucine rich motifs, which bind directly to the ligand-binding domain (LBD) in ER. NF1 has several of these motifs that are much more highly conserved in species with a functional ER pathway, and some of these are mutated in cancers (e.g., in our patient cohort). Furthermore, we found that NF1 canbind directly to ER, and that this binding is mediated between the ER LBD and the NF1 leucine-rich regions. Like a classic co-repressor, wildtype NF1 (but not mutants lacking GAP activity or the Leu-rich motif) binds to ER, and is recruited by ER to the ERE in the presence of tamoxifen, but not E2. Further preclinical treatment studies indicate that while NF1-deficient ER+ breast cancer should not be treated by tamoxifen or AIs, fulvestrant remains effective. Furthermore, when fulvestrant is combined with dabrafinib and trametinib to inhibit Ras effectors Raf and MEK, apoptosis is induced in vitro, and tumor regression is observed in vivo. In conclusion, we have demonstrated that NF1 is a dual negative regulator at the intersection of two potent oncogenic signaling pathways, Ras and ER, and that NF1-deficient ER+ breast cancer patients may be more effectively treated by co-targeting the Ras and ER signaling. These patients, up to 10% of those with advanced ER+ breast cancer, can be readily identified for treatment by ctDNA analysis. A clinical trial is under development. Citation Format: Chang EC, Zheng Z, Philip L, Burcu C, Lei J, Singh P, Anurag M, Chan D, Li JD, Du XP, Shafaee MN, Banks K, Sacker S, Song W, Nguyen T, Cao J, Chen X, Haricharan S, Kavuri M, Kim B-J, Zhang B, Gutmann DH, Lanman RB, Foulds C, Ellis M. Direct regulation of estrogen receptor-α (ER) transcriptional activity by NF1 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS2-02.
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