Cutaneous squamous cell carcinoma (cSCC) is a prevalent malignancy arising from keratinocytes, representing the second most common form of non-melanoma skin cancer [1]. The global age-standardized incidence of cSCC is approximately 30.30 per 100,000 and has been steadily increasing in recent years, posing a growing public health challenge [2]. Surgical excision remains the mainstay for most localized cSCC cases. For locally advanced or metastatic disease, available therapeutic options include radiotherapy, chemotherapy (e.g., cisplatin and 5-fluorouracil), targeted therapy with EGFR inhibitors, and immune checkpoint inhibitors [3]. However, the clinical efficacy of these systemic treatments is often limited by dose-limiting toxicities, short-lived responses, and contraindications in high-risk groups such as organ transplant recipients [4]. Consequently, there is an imperative need to decipher the underlying molecular mechanisms of cSCC and identify targets for novel therapeutic interventions.

One‑carbon metabolism is an intricate network of biochemical reactions responsible for the uptake, transfer, and utilization of one‑carbon units, which are critical for nucleotide biosynthesis, epigenetic modifications, and maintenance of redox homeostasis [5]. A pivotal enzyme in this pathway is methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), which catalyzes the conversion of 5,10-methylene-tetrahydrofolate to 10-formyl-tetrahydrofolate, concurrently generating NAD(P)H [6]. MTHFD2 is abundantly expressed during embryogenesis but markedly downregulated in most healthy adult tissues. Nonetheless, it ranks among the most frequently upregulated metabolic enzymes across diverse cancer types [7]. MTHFD2 has been shown to facilitate the progression of multiple malignancies, such as acute myeloid leukemia [8], glioblastoma [9], lung cancer [10], hepatocellular carcinoma [11], gastric cancer [12], esophageal cancer [13], colorectal cancer [14], renal cell carcinoma [15] and prostate cancer [16], through its canonical metabolic functions or non-metabolic mechanisms [6]. MTHFD2 orchestrates several key tumor biological processes, such as redox homeostasis, epigenetic regulation and immune modulation [12], [15], [17]. However, its biological role and underlying mechanisms in cSCC remain largely unclear.

In addition to one‑carbon metabolism, cancer cells activate a broader metabolic reprogramming that encompasses dysregulated glucose and amino acid metabolism, as well as enhanced de novo lipogenesis [18]. This lipogenic switch is critical for meeting the heightened cellular demands for membrane biogenesis, energy reservoir, and lipid signaling in rapidly proliferating tumor cells [19]. A pivotal enzyme in this process is fatty acid synthase (FASN), which orchestrates the de novo synthesis of long-chain fatty acids. Despite its minimal presence in most normal tissues, FASN is universally observed to be overexpressed during neoplastic transformation, with its levels strongly predicting tumor progression and poor clinical outcomes [20].

In this study, we evaluated MTHFD2 expression in clinical cSCC specimens and cell lines. Using both gain- and loss-of-function approaches, we then investigated the functional consequences of MTHFD2 on cSCC cell proliferation, apoptosis, migration, and invasion. Additionally, we conducted metabolic analyses, RNA sequencing, and immunoprecipitation-mass spectrometry to deeply explore the mechanisms. Our findings first demonstrate that MTHFD2 reprograms cellular metabolism and interacts with FASN, activating a FASN/PI3K-AKT signaling axis, thereby driving cSCC progression. This study uncovers a dual metabolic and non-canonical oncogenic function of MTHFD2 in cSCC, highlighting its potential as a new therapeutic target for cSCC treatment.