Effects of folic acid withdrawal on transcriptomic profiles in murine triple-negative breast cancer cell lines

Highlights

• Folic acid withdrawal alters gene expression in mouse mammary cancer cell lines.

• Changes are most pronounced in non-metastatic mesenchymal cells.

• Interferon signaling pathway genes are upregulated by folic acid withdrawal.

• Reactivation of the type I interferon pathway may have clinical utility.

Abstract

We have previously shown that withdrawal of folic acid led to metabolic reprogramming and a less aggressive phenotype in a mouse cell model of triple-negative breast cancer (TNBC). Herein, we evaluate the effects of folic acid withdrawal on transcriptomic profiles in these cells. Murine cell lines were originally derived from a pool of spontaneous mammary tumors grown in MMTV-Wnt1 transgenic mice. Based on their differential molecular characteristics and metastatic potential, these cell lines were previously characterized as non-metastatic epithelial (E-Wnt), non-metastatic mesenchymal (M-Wnt) and metastatic mesenchymal (metM-Wnt^liver) cells. Using custom two-color 180K Agilent microarrays, we have determined gene expression profiles for three biological replicates of each subtype kept on standard medium (2.2 μM folic acid) or folic acid-free medium for 72 h. The analyses revealed that more genes were differentially expressed upon folic acid withdrawal in M-Wnt cells (1884 genes; Benjamini-Hochberg-adjusted P-value <0.05) compared to E-Wnt and metM-Wnt^liver cells (108 and 222 genes, respectively). Pathway analysis has identified that type I interferon signaling was strongly affected by folic acid withdrawal, with interferon-responsive genes consistently being upregulated upon folic acid withdrawal in M-Wnt cells. Of note, repressed interferon signaling has been established as one of the characteristics of aggressive human TNBC, and hence reactivation of this pathway may be a promising therapeutic approach. Overall, while our study indicates that the response to folic acid withdrawal varies by molecular subtype and cellular phenotype, it also underscores the necessity to further investigate one-carbon metabolism as a potential therapeutic means in the treatment of advanced TNBC.

Introduction

Cancer metastasis is the most common cause of cancer-related death [[1], [2], [3]]. Metastasis is a complex multi-step process, with cells undergoing metabolic reprograming to support their energetic, biosynthetic, and redox needs at various stages of metastasis [[4], [5], [6]]. During invasion, metabolic adaptation also enables cancer cells to survive in nutrient-limited environments [5,6]. Epithelial-to-mesenchymal transition (EMT), a process where epithelial cancer cells acquire mesenchymal features, plays an important role in cancer cell invasion and metastasis [6,7]. EMT is regulated by several transcription factors, which control, among others, numerous metabolic genes [5,6].

One of the key metabolic branches in the cell is the network of folate-dependent biochemical pathways. Folate (vitamin B9) functions as a coenzyme in numerous reactions of one-carbon transfer, which are involved in nucleotide biosynthesis, amino acid metabolism, redox homeostasis, and epigenetic regulation [[8], [9], [10]]. Since humans cannot synthesize folate, folate metabolism is regulated to a significant extent by dietary intake of the vitamin. Low folate intake or status has been associated with an increased risk of certain diseases, most notably neural tube defects [[10], [11], [12]], but also particular forms of cancer [13,14]. In 1996, the U.S. Food and Drug Administration (FDA) approved mandatory fortification of several types of grain foods in the US with folic acid, a synthetic form of the vitamin, to prevent neural tube defects [15].

Replenishing the intracellular folate pool by the intake of exogenous folates, such as folic acid, is especially important for rapidly dividing cells, including cancer cells. Ample preclinical evidence links cellular replication to folate availability [[16], [17], [18], [19], [20], [21], [22], [23]]. Such a link raised the concern that excessive consumption of dietary folate or folic acid may promote the progression of neoplastic lesions and increase the risk of malignancies and cancer-related death [11,[24], [25], [26]]. In support of such concern, animal studies have demonstrated that high intake of folic acid promotes the progression of established tumors [[27], [28], [29], [30], [31]]. A similar conclusion has been drawn based on the observed association between serum folate levels and proliferation of cancer cells in men with prostate cancer [32]. Of note, while additional studies support this conclusion [33], other studies have shown inverse associations between dietary folate and tumor development [34]. Nevertheless, the possibility that excessive intake of folates might promote development of malignancies should not be ignored [13,35,36].

The role of folate in cancer metastasis is even less clear and the literature on this subject is limited and somewhat controversial. Low extracellular folate is associated with altered expression of genes involved in cell adhesion, migration and invasion [[37], [38], [39]] indicating that the vitamin might play a role in the mechanism of metastasis. Of note, the positive association between folic acid supplementation and the migratory ability of cancer cells, as well as the inhibitory effects of antifolates on cell migration, have been demonstrated [[40], [41], [42]]. In further support of the role of folate metabolism in metastasis, metastasizing melanomas have been shown to have increased dependence on NADPH-generating folate pathways [43]. Also, antifolate pemetrexed has been approved by the FDA for the treatment of advanced or metastatic non-small cell lung cancer [[44], [45], [46]]. Several studies, however, suggested that folate supplementation would inhibit rather than promote metastasis. For example, a recent report has demonstrated that in cultured cells folate deficiency causes the elevation of reactive oxygen species, which leads to the suppression of proliferation, but at the same time enhances metastatic potential [47]. In agreement with these findings, folate deficiency promoted metastasis in a xenograft mouse model [47]. In another study, folate deprivation enhanced invasiveness of colon cancer cells through the activation of sonic hedgehog and NF-κB signaling [48]. Inhibitory effects of folic acid on metastasis and proliferation have been also linked to the activation of the folate receptor-ERK1/2-TSLC1 signaling pathway [49]. Contrary to these studies, we have previously reported that folate deprivation inhibits migration and invasion of cultured cancer cells and decreases tumor growth and metastasis in a mouse model [28,42,50].

In a recent study, we have shown that folic acid restriction led to metabolic reprogramming and a less aggressive phenotype in cultured murine triple-negative breast cancer (TNBC) cells [50]. Specifically, cells kept on folic acid-free medium displayed reduced migration and had weaker invasion potential [50]. Interestingly, effects of folic acid withdrawal were more profound in more tumorigenic mesenchymal cells than in epithelial cells [50]. To obtain insight into potential mechanisms related to folate restriction, in the present study we have evaluated transcriptomic profiles in three mouse TNBC cell lines grown in medium with or without folic acid.