Mitochondrial MTHFD isozymes display distinct expression, regulation, and association with cancer

Highlights

• Mammalian cells possess two mitochondrial MTHFD isozymes, MTHFD2 and MTHFD2L.

• Both isozymes are present in normal and cancer cells, but MTHFD2 has in general higher expression.

• MTHFD2 displays a more prominent response to growth factor stimulation.

• There is no compensatory increase of MTHFD2L following inhibition of MTHFD2.

• The therapeutic targeting of mitochondrial folate pathway should focus on the MTHFD2 isozyme.

Abstract

Mitochondrial folate metabolism is central to the generation of nucleotides, fuelling methylation reactions, and redox homeostasis. Uniquely among the reactions of the mitochondrial folate pathway, the key step of the oxidation of 5,10-methylene-tetrahydrofolate (CH₂-THF) can be catalysed by two isozymes, MTHFD2 and MTHFD2L. The MTHFD2 enzyme has recently received considerable attention as an oncogenic enzyme upregulated in several tumour types, which is additionally required by cancer cells in vitro and in vivo. However, much less is currently known about MTHFD2L and its expression in cancer. In this study, we examine and compare the expression and regulation of the two mitochondrial MTHFD isozymes in normal human and cancer cells. We found that normal and cancer cells express both enzymes, although MTHFD2 has a much higher baseline expression. Unlike MTHFD2, the MTHFD2L isozyme does not show an association with proliferation and growth factor stimulation. In addition, we did not find evidence of a compensatory increase of MTHFD2L following suppression of its isozyme. This study supports that MTHFD2L is unlikely to have an important function in increased proliferation or cancer. Furthermore, therapeutic strategies aiming to block the mitochondrial folate pathway in cancer should focus on MTHFD2, with MTHFD2L being unlikely to be involved in the development of chemoresistance to targeting of its mitochondrial isozyme.

Introduction

Folate mediated one-carbon (1C) metabolism supports essential biological processes, which are often deregulated in cancer cells, such as biosynthesis of purines and thymidylate, homeostasis of amino acids (serine, glycine, methionine), methylation reactions, and regeneration of anti-oxidants. In eukaryotic cells, the folate 1C metabolism is compartmentalised into cytoplasmic, nuclear, and mitochondrial pathways (Tibbetts and Appling, 2010; Ducker and Rabinowitz, 2017). Experimental evidence supports that the mitochondrial compartment of the folate 1C pathway is the source for the vast majority of 1C units. These 1C units originate from molecules such as serine and glycine, and are processed through the folate pathway to generate formate units, which are eventually released into the cytosol (MacFarlane et al., 2008; Pike et al., 2010; Ducker et al., 2016).

Within the mitochondrial 1C pathway, each step is catalysed by a single enzyme, with the exception of the presence of two distinct bifunctional CH₂-THF dehydrogenase/CH⁺-THF cyclohydrolase enzymes, MTHFD2 and MTHFD2L (Fig. 1). These two isozymes catalyse the conversion of CH₂-THF into CHO-THF, which is the substrate for the production of formate through the action of MTHFD1L. Of these two isozymes, the most studied by far is MTHFD2. This enzyme was initially identified as a NAD⁺-dependent 5,10-CH₂-THF dehydrogenase in 1985 (Mejia and MacKenzie, 1985). In recent years, a number of studies have implicated this enzyme in cancer. In a comparison of 14 different tumour/normal tissues, it was found that MTHFD2 is the most upregulated out of >1000 metabolic enzymes examined (Nilsson et al., 2014). Inhibition of MTHFD2 has been reported to affect proliferation and survival of various cancer cells, including lung (Nishimura et al., 2019), breast (Lehtinen et al., 2013; Koufaris et al., 2016a), and acute myeloid leukemia (Pikman et al., 2016). Consequently, MTHFD2 is considered a novel and promising therapeutic target, with a number of efforts in developing MTHFD2 inhibitors already reported (Fu et al., 2017; Gustafsson et al., 2017; Asai et al., 2018).

In contrast, the second mitochondrial MTHFD isozyme, MTHFD2L, has, so far, received much less attention. The Appling group has been the driver behind the identification and characterisation of this second mitochondrial MTHFD isozyme. By searching for novel vertebrate genes with sequence similarity to MTHFD1, they were able to identify and clone the MTHFD2L enzyme. It was also demonstrated that this enzyme possesses CH₂-THF activity and that it is expressed in various adult tissues (Bolusani et al., 2011). In a subsequent study, the same group tested the isolated protein to verify that it had the same bifunctional catalytic capabilities as MTHFD2. Gene expression profiling in adult mice tissues found that the mRNA levels of MTHFD2L were much lower than MTHFD2, with the exception of the testes and spleen (Shin et al., 2014). Finally, in a more recent paper, this group showed that, besides possessing similar bifunctional enzymatic capabilities, both enzymes can use NAD+ or NADP+ as cofactors (Shin et al., 2017).

Despite the increasing attention given to the mitochondrial folate pathway in recent years, the interaction and roles of the MTHFD2/MTHFD2L dyad are still not understood. For instance, it has not yet been clarified whether cancer cells inverse the expression of the two isozymes, from low MTHFD2/high MTHFD2L to the reverse, and whether only expression of MTHFD2 is increased or whether both display increased levels. Another open issue is whether, upon suppression of MTHFD2, cells respond by increasing the levels of the MTHFD2L isozyme. Beyond biological interest, these questions are also of significant importance for optimising drug design strategies, e.g. whether chemicals targeting just MTHFD2 or both isozymes would be more efficient for targeting cancer cells.

In this study, we used a combination of experimental methods and the examination of publicly available data to compare, contrast, and define the regulation and expression of the two mitochondrial MTHFD isozymes. First, using this combinatorial approach, we show that the MTHFD2L isozyme is less dynamically regulated compared to MTHFD2, supporting a more baseline or housekeeping function. Second, cancer cells retain expression of both isozymes, but only MTHFD2 displays prominent upregulation in cancer. Third, MTHFD2L is not involved in the adaptive response of cells to suppression of MTHFD2. The novel insights generated by this project are important for understanding the regulation and function of the mammalian folate pathway.