An novel inhibitor of TGF-β type I receptor, IN-1130, blocks breast cancer lung metastasis through inhibition of epithelial–mesenchymal transition
Introduction
Breast cancer is the most common malignant disease that affects females worldwide [1]. Most cancer deaths are caused by the formation of metastasis rather than by the primary tumor [2].
Metastasis is multi-step process, and one mechanism associated with tumor cell invasion and metastasis is the induction of epithelial–mesenchymal transition (EMT), which is characterized by reduced cell–cell adhesion and by increased cell motility [3]. Previous studies have demonstrated that canonical TGF-β/Smad signaling plays a pivotal role in EMT in vitro and in vivo [4], [5], [6]. Thus, the inhibition of the TGF-β/Smad signaling pathway offers a rational approach for cancer therapy. Thus far, three approaches have been used to inhibit TGF-β/Smad signaling: antisense oligonucleotides [7], [8], monoclonal antibodies [9], [10], and chemical inhibitors. Small-molecule inhibitors of TGF-β/ALK5 kinase activity, such as SD-208 [11] and LY-2157299 [12] have been successfully used to suppress tumor development and metastasis in animal models. Nonetheless, there are no available anti-TGF-β therapies for patients with metastatic cancer.
To develop anti-TGF-β therapeutics, we synthesized a novel small-molecule ALK5 inhibitor, IN-1130, and investigated its potential as an anti-TGF-β therapy. We found that IN-1130 inhibited TGF-β/Smad signaling, cell migration, invasion, and lung metastasis in 4T1 orthotopic xenograft mice and in MMTV/c-Neu transgenic mice. IN-1130 increased the survival of mice bearing 4T1 breast cancer. Taken together, these data suggest that IN-1130, a small-molecule ALK5 inhibitor, could be developed as an anti-cancer therapeutic against breast cancer metastasis to the lungs.
Section snippets
Reagents and plasmids
Human recombinant TGF-β1 was purchased from R&D. IN-1130, SB-505124, p3TP-Lux, and pSBE-Luc have been previously described [13], [14], [15]. To construct the pGL4(CMV)-Luc vector, the CMV promoter was amplified, and the resulting PCR products were inserted into the pGL4.17[luc2/Neo] vector (Promega).
Cell lines and cultures
MDA-MB-231 cells were cultured in RPMI-1640, and HaCaT, HepG2, NMuMG, and 4T1 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). MCF10A cells were maintained as described
IN-1130 inhibited in vivo breast cancer metastasis to the lungs in MMTV/c-Neu mice
We examined the effect of IN-1130 on the metastasis of breast tumors to the lungs using breast tumor-bearing MMTV/c-Neu female transgenic mice treated with IN-1130 (40 mg/kg) via intraperitoneal injection 3 times per week for 3 weeks. IN-1130-treated mice had decreased breast cancer metastasis to the lungs compared with untreated MMTV/c-Neu mice (Fig. 1A), but there were no changes in the primary breast tumor volume or body weight (data not shown). These results were consistent with previous
Discussion
TGF-β is a ubiquitously expressed cytokine that regulates various cell behaviors, including cell proliferation, differentiation, migration, apoptosis, and wound healing [19], [20]. The binding of TGF-β ligand to TGF-β type II receptor (TβRII) leads to the formation of a heterotetrameric complex with a TGF-β type I receptor (TβRI or ALK5), which results in the phosphorylation of TβRI by TβRII [21]. Activated TβRI propagates signaling by recruiting and phosphorylating Smad2 and Smad3 at the
Conflict of Interest
No potential conflicts of interests were disclosed.
Grant support
This work was supported by a Korea Science and Engineering Foundation (KOSEF) Grant funded by the Korea government (MEST) (No. 20090093972).
Acknowledgments
The authors thank Dr. Sri Ram (NIH/Retired) and Dr. Lalage M. Wakefield (NIH) for critical review and discussion of the manuscript, and Min-Kyung Park and Jungin Jee for maintaining the animals and collecting tissue samples.
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