Gremlin-2 is a novel tumor suppressor that negatively regulates ID1 in breast cancer.
Breast cancer is one of the most common cancers in women and is closely associated with obesity. Gremlin-2 (GREM2), an antagonist for bone morphogenetic proteins (BMPs), has been considered an inhibitor of adipogenic differentiation in adipose-derived stromal/stem cells. However, the role of GREM2 in breast cancer cells remains largely unknown, and its signaling mechanism has yet to be clarified.
Bioinformatics analysis was conducted using public databases. Breast cancer cells overexpressing mock or GREM2 were used for in vitro and in vivo studies. Cell viability, colony formation, migration, and animal studies were performed to investigate the role of GREM2 in breast cancer cells. Screening of target genes affected by GREM2 overexpression in breast cancer cells was performed through RNA sequencing (RNA-seq) analysis.
The expression level of GREM2 mRNA was significantly reduced in both breast cancer tissues and cell lines. Kaplan-Meier analysis showed that low expression of GREM2 and high methylation of the GREM2 promoter were each associated with poor patient survival. The low mRNA expression of GREM2 in breast cancer cells was increased by the demethylating agent decitabine. Breast cancer cells overexpressing GREM2 decreased cell proliferation when compared to control cells, both in vitro and in vivo. Through comparison of RNA-seq analysis between cell lines and tissue samples, gene ontologies that were consistently upregulated or downregulated by GREM2 in breast cancer were identified. In particular, the expression of inhibitor of DNA-binding-1 (ID1) was repressed by GREM2. BMP2 is one of the upstream regulators that increases the expression of ID1, and the expression of ID1 reduced by GREM2 was restored by overexpression of BMP2. Also, the migration ability of breast cancer cells, which had been suppressed by GREM2, was restored by BMP2 or ID1.
Low expression of GREM2 in breast cancer cells is associated with hypermethylation of the GREM2 promoter, which may ultimately contribute to poor patient survival. GREM2 participates in regulating the expression of various genes, including ID1, and is involved in suppressing the proliferation of breast cancer cells. This suggests that GREM2 has the potential to act as a novel tumor suppressor in breast cancer.
Jung J
，Kim NH
，Park J
，Lim D
，Kwon M
，Gil W
，Jung S
，Go M
，Kim C
，Cheong YH
，Lee MH
，Park HS
，Eom YB
，Park SA
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Protective effects of miR-24-2-5p in early stages of breast cancer bone metastasis.
Bone is the most frequent site of metastasis for breast cancer (BC). Metastatic BC cells interact with bone cells, including osteoclasts and osteoblasts, creating a cancer niche where they seed and proliferate. MicroRNAs (miRNAs) are regulators of breast-to-bone metastasis progression. MiR-24-2-5p has previously been shown to have roles in both breast cancer progression and inhibition of osteogenic differentiation. However, a direct link between miR-24-2-5p activity and the onset of bone metastasis remains ill-defined.
Analysis of the expression of miR-24 forms (miR-24-2-5p, miR-24-3p, miR-24-1-5p) in the serum from early-stage BC patients at baseline (time of surgery) was conducted. MiR-24-2-5p overexpression in BC cells (NW1, a luc2-positive subpopulation of MDA-MB-231, and MCF7) was obtained by miRNA mimic transfection or lentivirus transduction. MiR-24-2-5p downregulation in BC cells (ZR-75-1, T-47D, SK-BR-3) was obtained by miRNA inhibitor transfection. Cell proliferation, migration and/or invasion assays were performed to assess BC cell functions after modulation of miR-24-2-5p expression. An animal model was used to assess the effect of miR-24-2-5p overexpression on early BC metastasis formation, as judged by bioluminescence imaging, and on bone remodelling, following measurement of circulating bone resorption (CTX-I) and bone formation (P1NP) markers. The effect of conditioned medium from miR-24-2-5p-overexpressing BC cells on human and murine osteoclast differentiation was investigated. Endogenous miR-24-2-5p expression levels were also quantified during murine osteoclast differentiation. RNA-sequencing (RNA-seq) analysis of BC cells was performed to evaluate transcriptomic changes associated with miR-24-2-5p overexpression. Selected modulated transcripts upon miR-24-2-5p overexpression were further validated by real-time qPCR.
Low expression levels of miR-24-2-5p, but not other miR-24 forms (miR-24-3p, miR-24-1-5p), in the serum from early-stage BC patients were associated with a high risk to develop future (bone) metastases. MiR-24-2-5p was also present in small extracellular vesicles secreted from BC cells. Forced expression of miR-24-2-5p in BC cells (NW1, MCF7) reduced their malignant traits (migration, invasion, and proliferation) in vitro. Furthermore, miR-24-2-5p overexpression in NW1 cells reduced metastasis, particularly in bone, and decreased bone turnover in vivo. RNA-seq and real-time qPCR analyses of NW1 and MCF7 cells overexpressing miR-24-2-5p showed the downregulation of common transcripts (CNNM4, DCTD, FMR1, PIGS, HLA-A, ICK, SH3BGRL2, WDFY, TRAF9B, IL6ST, PEX10, TRIM59). The conditioned medium from BC cells overexpressing miR-24-2-5p decreased human and murine osteoclast differentiation in vitro. Additionally, endogenous miR-24-2-5p expression levels in murine bone marrow-derived monocytes decreased during their differentiation into osteoclasts, further suggesting an inhibitory role for miR-24-2-5p during osteoclastogenesis.
MiR-24-2-5p exerts multiple protective roles in the early steps of BC bone metastasis by reducing malignant BC cell traits and tumour cell dissemination in bone, as well as by reducing the differentiation of precursors into mature osteoclasts.
Puppo M
，Croset M
，Ceresa D
，Valluru MK
，Canuas Landero VG
，Hernandez Guadarrama M
，Iuliani M
，Pantano F
，Dawn Ottewell P
，Clézardin P
... -
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NOSH-aspirin (NBS-1120) inhibits estrogen receptor-negative breast cancer in vitro and in vivo by modulating redox-sensitive signaling pathways.
Estrogen receptor (ER)-negative breast cancers are known to be aggressive and unresponsive to antiestrogen therapy, and triple-negative breast cancers are associated with poor prognosis and metastasis. Thus, new targeted therapies are needed. Forkhead box M1 (FOXM1) is abundantly expressed in human cancers and implicated in protecting tumor cells from oxidative stress by reducing the levels of intracellular reactive oxygen species (ROS). Aspirin, a prototypical anticancer agent with deleterious side effects that has been modified to release nitric oxide and hydrogen sulfide is called nitric oxide-hydrogen sulfide-releasing aspirin (NOSH-aspirin, NOSH-ASA), generating a "safer" class of new anti-inflammatory agents. We evaluated NOSH-ASA against ER-negative breast cancer using cell lines and a xenograft mouse model. NOSH-ASA strongly inhibited growth of MDA-MB-231 and SKBR3 breast cancer cells with low IC50s of 90 ± 5 and 82 ± 5 nM, respectively, with marginal effects on a normal breast epithelial cell line. NOSH-ASA inhibited cell proliferation, caused G0/G1 phase arrest, increased apoptosis, and was associated with increases in ROS. In MDA-MB-231 cell xenografts, NOSH-ASA reduced tumor size markedly, which was associated with reduced proliferation (decreased proliferating cell nuclear antigen expression), induction of apoptosis (increased terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells), and increased ROS, whereas nuclear factor κ-light-chain-enhancer of activated B cells and FoxM1 that were high in untreated xenografts were significantly reduced. mRNA data for FoxM1, p21, and cyclin D1 corroborated with the respective protein expressions and arrest of cells. Taken together, these molecular events contribute to NOSH-ASA-mediated growth inhibition and apoptotic death of ER-negative breast cells in vitro and in vivo. Additionally, as a ROS inducer and FOXM1 inhibitor, NOSH-ASA has potential as a targeted therapy. SIGNIFICANCE STATEMENT: We examined the cellular effects and xenograft tumor inhibitory potential of NOSH-aspirin, a nitric oxide- and hydrogen sulfide-donating hybrid, against estrogen receptor-negative breast cancer, which currently lacks effective therapeutic options. Inducing reactive oxygen species and downregulating forkhead box M1 are plausible mechanisms contributing to decreased cell proliferation and increased apoptosis. NOSH-aspirin reduced tumor size by 90% without inducing any observable gross toxicity, underscoring its promising translational potential.
Chattopadhyay M
，Nath N
，Kodela R
，Metkar S
，Soyemi SA
，Kashfi K
... -
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ResearchGate
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