Cancer Letters

Cancer Letters

Volume 351, Issue 1, 28 August 2014, Pages 72-80
Cancer Letters

An novel inhibitor of TGF-β type I receptor, IN-1130, blocks breast cancer lung metastasis through inhibition of epithelial–mesenchymal transition

https://doi.org/10.1016/j.canlet.2014.05.006Get rights and content

Highlights

  • IN-1130 inhibited Smad/TGF-β signaling, EMT, cell migration, and invasion.

  • IN-1130 inhibited lung metastasis of mammary tumor in mice models.

  • IN-1130 prolonged the life span of tumor bearing mice.

Abstract

TGF-β signaling plays an important role in breast cancer progression and metastasis. Epithelial–mesenchymal transition (EMT) is an important step in the progression of solid tumors to metastatic disease. We previously reported that IN-1130, a novel transforming growth factor-β type I receptor kinase (ALK5) inhibitor, suppressed renal fibrosis in obstructive nephropathy (Moon et al., 2006). Here, we show that IN-1130 suppressed EMT and the lung metastasis of mammary tumors in mouse models. Treating human and mouse cell lines with IN-1130 inhibited TGF-β-mediated transcriptional activation, the phosphorylation and nuclear translocation of Smad2, and TGF-β-induced-EMT, which induces morphological changes in epithelial cells. Additionally, we demonstrated that IN-1130 blocked TGF-β-induced 4T1 mammary cancer cell migration and invasion. The TGF-β-mediated increase in matrix metalloproteinase (MMP)-2 and MMP-9 expression was restored by IN-1130 co-treatment with TGF-β in human epithelial cells and in 4T1 cells. Furthermore, we found that lung metastasis from primary breast cancer was inhibited by IN-1130 in both 4T1-xenografted BALB/c mice and MMTV/c-Neu transgenic mice without any change in primary tumor volume. IN-1130 prolonged the life span of tumor-bearing mice. In summary, this study indicated that IN-1130 has therapeutic potential for preventing breast cancer metastasis to the lung.

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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