The involvement of M2 macrophage polarization inhibition in fenretinide-mediated chemopreventive effects on colon cancer

Highlights

• 4-HPR could significantly suppress M2-like polarization of macrophages.

• The inhibition of phosphorylation of STAT6 is involved in the 4-HPR-driven inhibition of M2 polarization.

• The inhibition of M2 polarization in involved in 4-HPR-mediated chemoprevention.

Abstract

Clinical studies have shown that fenretinide (4-HPR) is an attractive chemopreventive agent for cancer treatment. However, to date, few studies have demonstrated the mechanism of the preventive effect of 4-HPR. In our current study, we revealed that 4-HPR could significantly suppress IL-4/IL-13 induced M2-like polarization of macrophages, which was demonstrated by the reduced expression of M2 surface markers, the down-regulation of M2 marker genes, and the inhibition of M2-like macrophages promoted angiogenesis. Mechanistically, our study suggested that the inhibition of the phosphorylation of STAT6, rather than the generation of oxidative stress, is involved in the 4-HPR-driven inhibition of M2 polarization. More intriguingly, by utilizing adenomatous polyposis coli (APC^min/+) transgenic mice, we demonstrated that the tumorigenesis was dramatically decreased by 4-HPR treatment accompanied with fewer M2-like macrophages in the tumor tissues, thereby profoundly blocking tumor angiogenesis. These findings, for the first time, reveal the involvement of M2 polarization inhibition in 4-HPR-mediated chemoprevention, which provides a new point of insight and indicates the potential mechanism underlying the chemopreventive effect of 4-HPR.

Introduction

Colorectal cancer (CRC), one of the most high-risk human malignant tumors, is the third most common cancer and the fourth leading cause of cancer deaths worldwide, accounting for approximately 1.2 million new cases and 0.6 million deaths per year [1]. Despite advances in the combined use of surgery, radiation, chemotherapy and other therapies, colorectal cancer patients still encounter several problems, such as recurrences, low postoperative 5-year survival rates and high metastasis rates. The 5-year relative survival for patients diagnosed with metastatic CRC is less than 15% [2]. Given that most cases of colorectal cancer are sporadic and develop slowly over a minimum of 5–10 years through the adenoma-carcinoma sequence, early detection and treatment are crucial for the improvement in morbidity and mortality of patients afflicted with this disease [3].

Tumors are composed of an array of cell types, including not only cancer cells but also the non-cancer cells. The most prominent component of these non-cancer cells are macrophages, which are called tumor-associated macrophages (TAMs) [4], [5]. Usually, monocytes or macrophages undergo profound functional reprogramming in response to various signals, cytokines, and the extreme outcomes of which reflect the plasticity of macrophages. M1/M2 polarized macrophages are extremes of a continuum in a universe of functional states. Classically activated macrophages (M1), alternatively activated macrophages (M2), or both have been observed in tumors [6]. It is generally considered that these two types of macrophages are functionally antagonistic. M2 macrophages are mainly involved in moderating inflammatory responses, promoting angiogenesis and contributing to tissue remodeling, all of which are, promote tumor progression [7], [8]. Increasing evidence suggests that some of the key molecules that are altered play significantly important roles in CRC progression and are associated with inflammation [9]. Moreover several investigations confirmed significantly higher levels of cathepsin B in CRC, which might be associated with the progression of colorectal adenoma to carcinoma [10], [11]. Of interest, evidence has shown that macrophages are the primary source of cathepsins [12]. Thus studying the relationship between macrophage infiltration and colorectal patients' tumor progression has attracted intensive interest, but this association has rendered conflicting results [13].

Fenretinide (4-HPR), a synthetic derivative of all-trans retinoic acid, can cause cancer cell death with the added benefit of having only minor side effects [14]. We and other groups have demonstrated that 4-HPR can induce apoptosis in multiple human cancer cell lines, both in vitro and in vivo [15], [16], [17]. In clinical trials, 4-HPR has been tested as both a chemopreventive agent in breast, bladder and oral mucosal cancers and a chemotherapeutic agent in pediatric and adult cancers [18], [19], [20]. Thus far, a possible benefit was only achieved in premenopausal women regarding the incidence of second breast malignancies of women with breast cancer, a result that persisted in a 15-year follow-up [18]. Given the chemopreventive effect of 4-HPR observed in the clinic, the mechanism of action of 4-HPR prevention of cancer incidence has drawn much attention [21], [22]. However, little progress has been made in this area as the molecular mechanisms underlying 4-HPR-mediated chemoprevention remain poorly understood.

In the current study, we observed that 4-HPR efficiently skewed macrophages away from M2 polarization induced by IL-4/IL-13 in vitro under a sub-cytotoxic concentration. Further, we demonstrated that the canonical pathway of STAT6 participated in the inhibition of the M2 polarization by 4-HPR. Additionally, by applying the APC^min/+ transgenic mouse model, we also showed that 4-HPR inhibited colorectal tumorigenesis in vivo through the prevention of tumor angiogenesis. Importantly, the in vivo effect of 4-HPR was linked to a decrease in the number of M2-like macrophages in the tumor region. Altogether, for the first time, our study showed that 4-HPR can inhibit TAM M2 polarization, which might explain the chemopreventive effect of 4-HPR in the clinic and open a new avenue to prevent CRC by applying modulators of TAM M2 polarization.