Multifaceted functions of transcription regulatory factor X6 (RFX6): from pancreatic development to cancer progression

Abstract

Regulatory factor X6 (RFX6) is defined as the sixth member of the RFX family based on its highly conserved and specific wing-helix DNA-binding domain. Its expression in adults is predominantly localized to pancreatic islets, small intestine, and colon. Extensive research has demonstrated that RFX6 regulates cellular processes, such as pancreatic development, differentiation of islet progenitor cells, and insulin secretion, through the modulation of specific miRNAs (such as miR145 and miR195) and mRNAs (such as Pdx1, Neurod1, GCK, and Abcc8). Hence, mutations and deletions in RFX6 have been linked to the onset of various types of diabetes, including type 2 diabetes, Maturity-onset diabetes of the young, neonatal diabetes mellitus, especially Mitchell-Riley Syndrome (MRS). Specifically, homozygous mutations in RFX6 impede the proper differentiation of pancreatic progenitor cells, leading to inhibition of pancreatic head-tail development and endocrine cell formation, thereby contributing to the pathogenesis of MRS. Furthermore, examination of RFX6 target genes reveals a potential association with tumor development, indicating that RFX6 may play a role in cancer progression. Dysregulated expression or mutations of the RFX6 gene in prostate cancer, hepatocellular carcinoma, gastric cancer, melanoma, and other tumors have garnered significant interest, with studies showing that such alterations affect tumor cell proliferation, migration, and invasion, and are correlated with an unfavorable clinical prognosis in patients carrying RFX6 mutations. This review delves into the various functions of RFX6, emphasizing its crucial regulatory roles in pancreatic development, tumorigenesis, and progression. In addition, recent advancements in MRS therapy are outlined, underscoring the importance of RFX6-targeted therapy in MRS and cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-025-04073-6.

Background

Regulatory factor X6 (RFX6) transcription factor belongs to the Regulatory factor X (RFX) family, which is widely distributed in eukaryotes, except in plants and algae [1, 2]. The DNA-binding domain (DBD) of RFX family members exhibits a high degree of conservation and specificity, playing a crucial role in facilitating the interaction of RFX transcription factors with the conserved cis-regulatory element known as the X-box motif [3]. Currently, there are eight documented members of the RFX family (RFX1-8) [4]. The RFX6 gene is situated on chromosome 6q22.1 and consists of 928 amino acids distributed across 19 exons [3]. Alongside the highly conserved DBD, the human RFX6 gene also features other conserved structural domains, such as the B, C, and dimerization domains [5]. The B and C domains, commonly known as the extended dimerization domains, facilitate the dimerization process [3]. In addition, RFX6 can interact with other members of the RFX family, mainly RFX2 and RFX3, to bind DNA as a dimer and play a regulatory role in the organism [3, 6]. However, RFX6 lacks a defined activation domain [4].

RFX transcription factors are widely expressed across a diverse array of tissues [4], with notable high expression in the immune system, gastrointestinal tract, reproductive system, and nervous system, where they play key roles in maintaining ciliary development and function [7–10]. In contrast, RFX6 exhibits a more restricted expression profile, being predominantly localized to the pancreas [6]. In murine models, RFX6 expression is initially observed in the terminal endoderm during embryonic development, then broadly expressed in the intestinal endoderm and becomes progressively restricted to the pancreatic buds and cells dispersed within the intestines during mid-gestation. Upon Neurog3 activation, RFX6 gene expression is observed in pancreatic progenitor cells, its expression is confined to the mature pancreas and islets of the intestinal tract and in later stages restricted to the islets of Langerhans in the mature pancreatic organ and the intestinal tract in mammals [6, 11]. In zebrafish, as in mice, RFX6 is present in pancreatic endocrine progenitor cells and mature islet cells, but it is not detected in the intestine [11]. In adult humans, RFX6 is primarily expressed in the pancreas and gastrointestinal tract, with lower levels of expression observed in the liver and heart [3, 6, 12]. These findings suggest that RFX6 plays a distinct role in pancreatic development.

In this paper, we have compiled and summarized the existing RFX6-related literature, and elaborated on RFX6 function with respect to pancreatic development, insulin secretion, Mitchell-Riley syndrome (MRS) treatment and tumor progression. These findings suggest the potential of RFX6 as a novel therapeutic target for diabetes and MRS, while also serving as a valuable reference for future research on the mechanisms of RFX6 action in tumors.

Key factor influencing pancreatic development and insulin secretion

The pancreas consists of distinct cell types with specialized functions. Endocrine cells—α-, β-, δ-, PP-, and ε-cells—regulate glucose through hormone secretion, while exocrine acinar and ductal cells produce digestive enzymes. Proper development of these populations is crucial for the pancreas to fulfill its essential roles in metabolism and digestion. In RFX6-deficient mice, notable observations included swollen bowel sounds attributed to small bowel obstruction, reduced pancreatic size, gastric heterotopia [13], impaired normal feeding, and early postnatal mortality [6]. Similarly, in RFX6-deficient zebrafish, pancreatic development was substantially disrupted, the differentiation of pancreatic endocrine progenitors was inhibited, and there was a significant decrease in the expression of genes related to endocrine function [12]. Notably, a significant suppression of Arx expression was observed in both RFX6-deficient mice and zebrafish [11, 12, 14]. The pancreatic islets comprise several endocrine cell types, with α-cells secreting glucagon to counterbalance insulin from β cells. Arx functions as a key determinant of α-cell fate by promoting glucagon gene expression and suppressing alternative endocrine lineages. Therefore, these findings indicate that RFX6 is positioned upstream of Arx and plays a crucial role in regulating α-cell differentiation and function within the pancreatic islets.

Studies have demonstrated that RFX6 does not bind to the Ins1 promoter, and in adult mice with RFX6 deficiency, the expression of key transcription factors involved in insulin production, including Mafa, Pdx1, and Nkx6.1, remains largely unchanged. Consequently, overall insulin production is not significantly altered, indicating that RFX6 is not essential for insulin production in adult mice [15]. In contrast, RFX6 deficiency during early developmental stages leads to defective pancreatic development and impaired insulin secretion, which are highly lethal. Thus, the functional requirement for RFX6 is stage-dependent, with a critical role in pancreatic development and β-cell differentiation, but only a minor role in maintaining insulin production in mature animals. Furthermore, a marked suppression of exocrine pancreatic gene expression was noted in Xenopus deficient in RFX6 [12]. In contrast, mice deficient in RFX6 exhibited no abnormalities in exocrine function [6], and zebrafish with RFX6 mutations showed no effect on the differentiation of exocrine cells [11]. Among patients with MRS, some exhibit severe exocrine dysfunction [16], whereas others remain unaffected [17]. Consequently, the impact of RFX6 deficiency and mutation on the exocrine pancreas remains unclear, warranting further investigation.

Importance of RFX6 in pancreatic development

In islet progenitor cells, RFX6 is activated by Neurog3 and is uniquely expressed in pancreatic islets, directing islet formation [6]. Neurog3 serves as a crucial transcription factor in facilitating the differentiation of β-cells and various other pancreatic islet cell types during embryogenesis [18, 19]. Inhibition of CDK-dependent phosphorylation of Neurog3 significantly increases the expression of downstream targets such as Neurod1, Insm1, Atoh8, and Rfx6, thereby promoting cellular differentiation [20]. RFX6 is co-expressed with Neurog3 in pancreatic islets [6, 21].However, RFX6 is not detectable in the pancreas of Neurog3-deficient mouse embryos [6, 22], whereas the absence of RFX6 does not affect Neurog3 expression [6], indicating that RFX6 acts downstream of Neurog3. Interestingly, RFX6 has been observed to partially restore islet development in the pancreas of Neurog3-deficient mice [23]. Nakamura et al. posited that Neurog3 deficiency may lead to more pronounced morphological abnormalities compared to RFX6 deficiency [24]. Neurog3 deficiency does not result in significant disruptions in the pancreas and intestines to the same extent as RFX6 deficiency, a finding that has been corroborated by studies conducted in both human and murine models [6, 25]. This indicates that the regulatory mechanism of RFX6 in the early endoderm is different from the mode dependent on Neurog3 activation in pancreatic endocrine cells.

Notably, a significant decrease in PDX1 expression was observed in RFX6-deficient mice and Xenopus [6, 12, 26]. PDX1 regulates pancreatic growth and development while acting in the transcriptional hierarchy controlling gastroduodenal functions [27, 28]. Chromatin immunoprecipitation sequencing analysis (ChIP-seq) confirmed that RFX6 is a direct target of PDX1 [29](Fig. 1). Furthermore, the analysis identified other pancreatic differentiation-related genes, including Neurod1, Hnf1a, Nkx6-1, St18, Shox2, and insulin translation-related genes Eif2ak1, Upf1, and Eif5, as targets of RFX6 [29]. The knockdown of RFX6 in postnatal zebrafish leads to a failure in insulin translation [30]. Additionally, miR-145 and miR-195 were identified by ChIP-seq as having RFX6 binding sites, which further modulate the process of pancreatic differentiation [29]. Collectively, these findings suggest that RFX6, functioning as a transcriptional regulator, persistently impacts pancreatic development by regulating target genes that govern both pancreatic differentiation and the insulin translation process.

Fig. 1.

RFX6 promotes pancreatic development and insulin secretion processes

In addition, an in vitro model of pancreatic differentiation utilizing human pluripotent stem cells identified two upstream microRNAs, miR-30d and let-7e, that target the expression of RFX6 [31]. However, during pancreatic development, the expression patterns of these microRNAs and RFX6 exhibit both correlation and anti-correlation, suggesting the involvement of multiple regulatory factors influencing this process [31].

Regulation of insulin secretion by RFX6

There was a marked reduction in the expression of transcription factors responsible for encoding insulin after RFX6 deletion [6]. In addition to pancreatic polypeptide cells, mice lacking RFX6 fail to form any other normal type of pancreatic islet cells, including β-cells [6]. β-cells are rare specialized cells that produce large amounts of insulin, and active insulin is stored in dense core secretory granules and is released via exocytosis thus regulating blood glucose levels [32]. RFX6 contributes to the maintenance of β-cell identity and function and normal islet morphology [33]. Piccand et al. suggested that RFX6 deficiency in adult β-cells causes glucose intolerance, impaired β-cell glucose sensing, and defective insulin secretion [15]. These conditions arise in association with reduced expression of core components of the insulin secretion pathway due to RFX6 deficiency, including glucokinase, the Abcc8/SUR1 subunit of the ATP-sensitive K⁺ channel (K_ATP channel), and voltage-gated Ca²⁺ channels (VDCCs) [15, 34]. GCK plays an important role in insulin secretion as well as glucose homeostasis [35]. GCK functions as a glucose sensor [36], linking blood glucose levels to metabolic signals within the β-cells [37], thereby modulating the ATP/ADP ratio, which in turn activates K_ATP channels and induces membrane depolarization. Abcc8/SUR1 encodes the regulatory sulfonylurea-binding subunit of the K_ATP channel, thereby connecting glucose metabolism to the electrical activity of the β-cells [38, 39]. Furthermore, genes responsible for the expression of VDCCs, such as Cacna1d and Cacna1c (L-type Ca²⁺ channels), Cacna1a (P/q-type Ca²⁺ channels), and Cacnb2 (β−2 subunit Ca²⁺ channels), influence Ca²⁺ influx and play a critical role in modulating β-cell insulin secretion [40, 41]. Studies have demonstrated that RFX6 directly regulates the transcription and expression of GCK and Abcc8, and also directly controls genes associated with VDCCs, specifically Cacna1c and Cacnb2 [34]. Consequently, these genes exhibit significant downregulation in pancreatic islets with deficient RFX6 expression [15, 34]. The absence of RFX6 expression results in reduced GCK levels, causing an imbalance in the ATP/ADP ratio. This imbalance, along with the downregulation of Abcc8/SUR1, modifies the excitability of β-cell membranes [37]. Concurrently, the inhibition of VDCCs results in impaired glucose-induced Ca²⁺ influx, ultimately causing defects in insulin secretion and thereby contributing to the development of diseases such as diabetes, etc [34].

In RFX6-inactivated adult β-cells, a group of so-called disallowed or forbidden genes (Ldha, Slc16a1, Pdgfra, Igfbp4, etc.) were found to be re-expressed [15]. The disallowed genes denote a group of genes that are typically expressed across various tissues but are selectively and profoundly repressed in adult β-cells [42]. Previous studies have indicated that certain disallowed genes, such as Ldha, influence insulin secretion upon expression [43]. Experimental evidence has demonstrated that RFX6 can directly bind to the conserved region of Ldha, thereby modulating its expression [15]. Consequently, we propose that RFX6 may regulate insulin secretion in conjunction with a diverse set of transcriptional regulators and contribute to this process through modulating the expression of ‘disallowed’ genes, such as Ldha. Furthermore, research utilizing the RFX6 defect model revealed a reduction in the expression of the zinc transporter Slc30a8, which is essential for normal insulin crystallization and secretion, thereby affecting the insulin secretion [6, 15]. The loss of RFX6 expression or the gain-of-function of disallowed genes can lead to impaired insulin secretion. Transcriptomic analyses have verified that RFX6 is an activated Nuclear factor 2 (NFATC2) dependent gene target, and NFATC2 is essential for maintaining RFX6 dependent β-cells differentiation and functional maturation [34]. Under conditions of metabolic and inflammatory stress, the Ca²⁺-dependent Calmodulin phosphatase (CN)/NFATC2 facilitates the recruitment of p300/HDAC1 to the promoters of the RFX6 and MCT1 genes [34]. This process induces the expression of RFX6 while inhibiting the transcription of the MCT1 (one of the disallowed genes) [44], thereby sustaining β-cell differentiation and modulating insulin secretion [34]. When there is excessive stimulation or depletion of Ca²⁺ signaling, CN/NFATC2 activity diminishes, leading to the suppression of RFX6 expression, induction of MCT1 expression, dedifferentiation of β-cells, and a marked reduction in insulin secretion [34].

Recent research indicates that RFX6 is highly expressed in intestinal endocrine K cells, with expression restricted exclusively to these cells [45, 46]. By constructing knockdown and overexpression models of RFX6, it has been demonstrated that RFX6 positively regulates the secretion of Gastric inhibitory peptide (GIP) and enhance GIP mRNA expression by binding to the active region of the GIP promoter [45]. GIP, also known as glucose dependent insulin-like peptide, is an intestinal insulinotropic protein produced by intestinal endocrine K cells located in the duodenum and upper small intestine, and functions to suppress gastric acid secretion and promote insulin secretion under glucose stimulation [47]. In summary, RFX6 can affect insulin secretion in K cells by regulating the expression of GIP.

A key player of RFX6 in diabetes mellitus and tumorigenesis

RFX6 and diabetes mellitus

MRS

Neonatal diabetes mellitus (NDM), defined as diabetes mellitus diagnosed within the first 6 months of birth, is an autosomal recessive syndrome in which pancreatic hypoplasia is the typical manifestation [48]. NDM exhibits a high degree of genetic heterogeneity, with various patterns of inheritance including dominant, recessive, and X-linked [49]. NDM is primarily classified into two forms: permanent neonatal diabetes mellitus (PNDM) and transient neonatal diabetes mellitus (TNDM) [50]. TNDM manifests as diabetes at birth, and enters a remission period several years later, usually progressing to T2D later in life [51]. PNDM lacks a remission phase and is typically divided into three types etiological types: abnormal pancreatic development, reduced β-cell mass, β-cell dysfunction [52]. Smith et al. were the first to identify a mutation in the RFX6 gene in patients with neonatal diabetes [6]. This particular case differs from the clinical manifestations of Martinez-Frias syndrome (MFS) and aligns with the symptoms documented by Mitchell et al. in 2004, leading to its initial designation as MRS [6]. The majority of MRS cases are classified under PNDM [53]. This study summarizes the currently identified RFX6 mutations associated with MRS (Fig. 2 and Table S1). Exons are labeled numerically, respectively, with all mutation sites in Maturity-onset diabetes of the young (MODY) at the top and all mutation sites in MRS at the bottom.

Fig. 2.

Mutation sites for RFX6 in MRS and MODY

MRS is caused by either homozygous or compound heterozygous mutations in the RFX6 gene [17]. MRS is believed supposed to be a variant of MFS [54] and has also been associated with severe inflammatory cholangiopathy in conjunction with other infantile cholestatic syndromes [55]. MRS is primarily characterized by neonatal diabetes, duodenal and jejunal atresia, pancreatic hypoplasia, and gallbladder hypoplasia [6, 17, 56]. In addition, some patients may present with cholestasis, refractory ascites, anemia, hemochromatosis, severe diarrhea, or gastric heterotopia, among other symptoms [16, 57–63]. The severity of these symptoms varies among individuals with MRS; however, the condition is generally associated with a high mortality rate, particularly during infancy.

In embryogenesis, a study revealed that RFX6 expression was significantly higher in the primitive gut tube (PGT) compared to the terminal endodermal stage [24]. During the PGT phase, PDX1 and SOX2 exhibit a high degree of co-expression with RFX6, and it has been confirmed that RFX6 can bind to regions upstream of the transcription start sites of PDX1 and CDX2 [24]. Therefore, RFX6 deletion resulted in decreased expression of the anterior-posterior and mid-posterior intestinal master regulators PDX1 and CDX2, but not the foregut marker SOX2 [24]. CDX2 is an intestinal-specific transcription factor involved in the regeneration and differentiation of intestinal epithelial cells and in maintaining the morphology and function of intestinal epithelial cells [64, 65]. Therefore, the diminished levels of PDX1 and CDX2 caused by RFX6 deficit could lead to pancreatic hypoplasia, intestinal atresia, diarrhea and gastric tissue ectopia in MRS [66–68]. In an experiment, induced pluripotent stem cells derived from individuals with MRS were cultivated in vitro and demonstrated normal differentiation capabilities during early developmental stages [24]. Nevertheless, following the generation of pancreatic endoderm, a significant impairment in differentiation was observed, with RFX6 deficiency specifically impairing the formation of endocrine cells in the head and tail regions of the pancreas, and resulting in the absence of the pancreatic body and tail [19, 69]. Given its role in regulating pancreatic progenitor cells and pancreatic development, homozygous mutations in RFX6 lead to pancreatic hypoplasia, as exemplified by the homozygous c.1129 C >T mutation identified in individuals with MRS [26]. Connective tissue resembling pancreatic tissue has been identified in the submucosa of the intestine in patients with RFX6-mutated MRS.

Several more effective treatments have been proposed for MRS in existing reported cases. For example, one approach involves surgically excising a portion of the ectopic gastric mucosa [53]. Gastric heterotopia occurs multiple times in MRS patients, with gastric mucosal ectopia in the small intestine resulting in malabsorption, chronic anemia, intestinal bleeding, and intestinal perforation [67, 70]. The patient in this case showed significant improvement in clinical symptoms at a later stage [53], suggesting that surgery may be an effective treatment modality for gastric ectopia in MRS. Moreover, individuals with MRS who receive Multi visual transplantation (MVT) early in life exhibit a positive prognosis characterized by near-normal gastrointestinal and pancreatic function, leading to one of the lengthiest post-treatment survival rates on record [68]. Consequently, testing the mucosa of the gastrointestinal tract as well as the structure and function of the pancreas in patients is now a more efficacious approach of treating as well as evaluating the condition of patients with MRS. Congenital glucagon-like peptide-1 (GLP-1) deficiency has been identified in certain cases of MRS. The GLP-1 receptor agonist liraglutide has demonstrated enhanced glycemic control and has significantly ameliorated abdominal pain, diarrhea, and other related symptoms [62]. These findings suggest that liraglutide may be a viable therapeutic strategy for managing neonatal diabetes resulting from RFX6 mutations.

MODY

MODY, which usually appears before the age of 25, is an autosomal dominant disorder [71], a form of monogenic diabetes caused by β-cell dysfunction [72]. Heterozygous mutations in transcription factors that play a role in the development and maturation of pancreatic β-cells are important in the development of MODY [73–75]. Fourteen MODYs have been identified, including GCK, HNF4A, HNF1A, HNF1B, PDX1, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, KCNJ11, and APPL1 [76–78]. In recent years, several truncating variants of the RFX6 protein have been identified within the MODY cohort [79]. This study summarized the existing RFX6 mutations in MODY, and the results are shown in Fig. 2. Heterozygous truncating variants of RFX6 have been linked to diminished penetrance and defects in GIP in MODY [79]. Most individuals with MODY associated with RFX6 mutations present with decreased pancreatic autoantibodies and reduced secretion of insulin, GIP, and GLP-1 [80, 81]. Consequently, GLP-1 agonists have demonstrated significant efficacy in enhancing glycemic control and are effective in the treatment of MODY. Furthermore, Dipeptidyl peptidase-4 (DPP-4) inhibitors have also been reported to elicit favorable therapeutic responses [72].

T2D

Type 1 diabetes mellitus is characterized by a complete deficiency of insulin resulting from the destruction of β-cells [82]. In contrast, Type 2 diabetes mellitus (T2D) is primarily attributed to a reduction in β-cell mass and dysfunction, leading to impaired insulin secretion [83, 84]. Nevertheless, the extent to which reduced β-cell mass and dysfunction contribute to the pathogenesis of T2D has been a subject of ongoing debate [85, 86]. Walker et al. employed a comprehensive, multimodal, and integrated methodology to assess pancreatic tissue and isolated islets from early T2D donors [87]. Their findings indicated that intrinsic defects in β-cell function are the predominant characteristic of this disease stage, while alterations in β-cell mass are not a significant factor. Further unbiased multimodal analyses identified a strong association between the RFX6 regulatory network and genetic risk for T2D, demonstrating that reduced expression of noncoding variants of RFX6 contributes to an elevated risk of developing T2D. RFX6 knockdown in β-cells induces transcriptional and chromatin changes. Genome-wide association studies have revealed that the regions of chromatin architectural changes caused by RFX6 overlap with common T2D variants. The changes are linked to the defective ion transport processes and dysregulated vesicle trafficking and exocytosis pathways mediated by changes in chromatin accessibility. Consequently, it is established that reduced expression of RFX6 in T2D contributes to compromised insulin secretion in β-cells. In mice exposed to a low-protein maternal diet leading to impaired development of fetal pancreatic islet β-cells, the body stimulates cell differentiation at the cost of proliferation through the upregulation of transcription factors such as RFX6 [88]. This ultimately results in a diminished reserve of β-cells and an elevated risk of developing T2D later in life.

Collectively, these findings underscore the critical role of RFX6 in preserving the functional and differentiated state of β-cells, suggesting that RFX6 may represent a novel therapeutic target for the treatment of T2D. Meanwhile, induction of the gene expression of RFX6 can be used to treat T2D with elevated blood glucose levels due to insufficient or resistant insulin secretion.

RFX6 and cancer

The RFX family has previously been demonstrated to be closely associated with cancer progression, with varying expression patterns observed across different types of cancer. Specifically, RFX1 functions as a transcriptional regulator that modulates the expression of proto-oncogenes and tumor suppressors [89]. In gastric [90], breast [91, 92], hepatocellular carcinoma [93–96], and esophageal cancers, among others, down-regulation of RFX1 expression has been observed. Overexpression of RFX1 has been shown to impede the epithelial-mesenchymal transition of cancer cells to reduce tumor invasion and metastasis [96]; additionally, increased RFX1 expression reduced the resistance to chemotherapy and cancer recurrence [92, 96]. Conversely, RFX1 exhibited a contrasting effect in metastatic ovarian cancer by facilitating metastasis upon overexpression [97]. Furthermore, heightened expressions of RFX3 [98] and RFX4 [99, 100] were observed in gliomas, contributing to glioma progression and impacting patient outcomes. Similarly, elevated levels of RFX5 [101] and RFX8 [102] were detected in cancerous tissues, promoting hepatocellular carcinoma advancement. Consequently, the involvement of RFX6 in cancer has garnered considerable attention in recent years.

Examination of pan-cancer data from the UCSC database revealed notable disparities in the expression profiles of RFX6 in cancerous tissues as opposed to normal counterparts (Fig. 3). A notable increase in RFX6 expression was observed in 7 tumor types, including Lung adenocarcinoma (LUAD), Esophageal carcinoma (ESCA), Prostate adenocarcinoma (PRAD), Liver hepatocellular carcinoma (LIHC), while a significant decrease was noted in 13 tumor types, such as Glioblastoma multiforme (GBM), Breast invasive carcinoma (BRCA), Colon adenocarcinoma (COAD), Stomach adenocarcinoma (STAD), Lung squamous cell carcinoma (LUSC), Thyroid carcinoma (THCA), Ovarian serous cystadenocarcinoma (OV), and Adrenocortical carcinoma (ACC). Through a review of existing literature, it was determined that RFX6 exhibits high expression levels in prostate cancer, hepatocellular carcinoma, and melanoma, while showing decreased expression in gastric cancer. These findings suggest that RFX6 may have varying roles as a transcription factor across different tumor types. Consequently, to provide a comprehensive overview of the role of RFX6 in cancer, the research advancements in each specific cancer type are outlined below.

Fig. 3.

RFX6 pan cancer expression analysis

Pan-cancer datasets standardized to TCGA, TARGET, and GTEx were obtained from the UCSC database by Sangerbox (Access Sangerbox free at http://www.sangerbox.com/tool.html). The expression levels of RFX6 in each sample were extracted and subjected to Log2 (X + 1) transformation. The expression differences between normal and tumor samples were calculated using R software (version 3.6.4) and analyzed for significance using unpaired Wilcoxon Rank Sum and Signed Rank Tests.

Prostate cancer

In a genome-wide association study of a Japanese male population, RFX6 was first identified as a susceptibility gene for prostate cancer [103], underscoring the significance of RFX6 in the context of cancer. Specifically, the study pinpointed a significantly associated susceptibility locus for prostate cancer, rs339331, situated on chromosome 6q22 within an evolutionarily conserved intronic region of the RFX6 gene [104]. Notably, the presence of the T risk allele at the SNP rs339331 locus exhibited a robust association with elevated RFX6 expression levels [105]. This finding was validated in both a Chinese male cohort and a black South African population [106–110], thereby confirming the link between genetic variation at the rs339331 locus and susceptibility to prostate cancer [101].

RFX6 is highly expressed in prostate cancer and is particularly upregulated when the tumor is in metastatic or advanced states [104]. Multiple sets of clinical data showed a positive correlation between the expression of HOXB13 and GATA2 with RFX6, suggesting that RFX6 may be a direct target of these transcription factors [111] (Fig. 4). HOXB13, a transcription factor with a homologous structural domain, is predominantly expressed in the adult prostate [112] and is significantly linked to androgen-dependent prostate cancer growth [113] and increased susceptibility to hereditary prostate cancer [114, 115]. The genetic variant rs339331 has been identified as being localized to prostate-specific enhancer elements upstream of the RFX6 gene, where it exhibits a preference for recruiting HOXB13 to the T risk allele [116]. This recruitment leads to increased binding of HOXB13 to transcriptional enhancers, followed by recruitment of AR and FOXA1 to the same region. This results in the formation of a transcriptional complex that promotes heightened expression of RFX6, ultimately increasing susceptibility to prostate cancer. The overexpression of GATA2 in prostate cancer is associated with increased cell proliferation, migration, and invasive capabilities, thereby facilitating tumorigenesis. In a similar manner, HOXB13 recruits GATA2 and SMAD4 to the RFX6 enhancers, forming a transcriptional complex that collectively influences the expression of the prostate cancer susceptibility gene RFX6. In summary, the rs339331 genetic variant promotes the recruitment of the transcription factor HOXB13 to RFX6 enhancers, leading to increased RFX6 expression, thereby facilitating the proliferation, migration, and invasion of prostate cancer cells and influencing various aspects of prostate cancer development, including onset, progression, metastasis, and biochemical recurrence [104].

Fig. 4.

Mechanisms by which RFX6 regulates tumor progression

Hepatocellular carcinoma

RFX6 is highly expressed in hepatocellular carcinoma, promotes cancer cell proliferation, migration, and invasion, and is associated with unfavorable patient prognosis [117]. Depletion of RFX6 triggers apoptosis in hepatocellular carcinoma cells and modulates tumor immune responses [118]. A study revealed a decrease in miRNA-542-3p expression in hepatocellular carcinoma, which was associated with the suppression of RFX6 expression [118]. RFX6 modulates hepatocarcinogenesis by targeting NOTCH1 and DTX2 proteins within the Notch signaling pathway as well as enhancing transcriptional stability. The Notch signaling pathway can facilitate tumor proliferation, invasion, and angiogenesis [119]. This indicates that the miRNA-542-3p-RFX6-DTX2-NOTCH1 regulatory pathway is significant in the progression of hepatocellular carcinoma [118]. Variations in the levels of glycerate mutase 1 (PGAM1) protein have the potential to impact the initiation of glycolysis [120]. Research has demonstrated that RFX6 promotes hepatocellular carcinoma growth and metastasis by transcriptionally regulating the expression level of PGAM1 and enhancing glycolysis [117]. Consequently, RFX6 is identified as a key regulator of glycolysis through the activation of its target genes, including PGAM1, ADH5, and GAPDH.

Melanoma

RFX6 is also a prognostic risk marker for melanoma, which contributes to 90% of skin cancer mortality. Six prognostically relevant genes, including RFX6, were screened by clinical, genomic, and transcriptomic data. Subsequent experiments confirmed that genes such as RFX6 were significantly overexpressed in tumor tissues compared to normal tissues, suggesting that RFX6 could be used as a new marker for the prognosis of melanoma patients [121].

Gastric cancer

Previous research has shown that homozygous mutations in RFX6 are associated with the development of MRS, a syndrome characterized by gastric heterotopia among other clinical symptoms. Valeria Calcaterra et al. concluded that patients with these mutations are at an increased risk of developing cancerous changes in the ectopic gastric mucosa and other tumor-related complications [67]. Analysis of transcriptomic data from databases indicated that alterations in RFX6 were the most notable among key transcription factors differentially expressed in gastric cancer [122]. Validation of these findings through clinical tissue analysis confirmed a significant downregulation of RFX6 expression in gastric cancer, suggesting that RFX6 could be a novel target for the treatment of gastric cancer.

Conclusion

Examination review on common scientific articles on the gene RFX6 suggests a main role in pancreatic development, MRS, and tumor progression. RFX6 is predominantly expressed in the pancreas and gastrointestinal tract, where it plays a crucial role in regulating pancreatic development, differentiation, and insulin translation by modulating the expression of downstream genes. Additionally, RFX6 is involved in the regulation of genes associated with β-cell maturation and function, ultimately impacting insulin secretion. Consequently, RFX6 abnormalities are likely to lead to diabetes, especially in MRS, which is typically characterized by pancreatic dysfunction and diabetes mellitus. Homozygous mutations in RFX6 are considered the main etiological factor. Nevertheless, the precise mechanisms underlying the occurrence of RFX6 mutations remain unclear. In the current phase of treatment investigation, primary therapeutic modalities for managing MRS include surgical excision of ectopic gastric mucosa or multimucosal transplantation during infancy. While advancements have been made in these treatment strategies, further clinical studies are required to validate the efficacy of therapeutic interventions and medications for MRS. RFX6 is anticipated to emerge as a significant target for MRS treatment in the foreseeable future.

Given the aberrant expression of RFX6 in numerous cancer types, it is imperative to elucidate the regulatory pathways of RFX6 in tumorigenesis. RFX6 exhibits varying expression across different tumor types, and this, coupled with its aberrant expression as well as upstream and downstream regulation affect tumor cell proliferation and cancer prognosis. The identification of RFX6 as a potential biomarker for predicting the recurrence or metastasis of cancer, as well as a promising therapeutic target, highlights its significance in the field of oncology. Furthermore, the latest study has revealed the involvement of RFX6 in glycolysis and its ability to modulate the expression of the pivotal protein PGAM1, thereby facilitating tumor advancement. This research has significantly advanced our comprehension of the mechanism of RFX6 action, offering a novel approach for cancer therapy. Examination of the upstream and downstream effects of RFX6 in both diabetes and tumorigenesis revealed a complex network of interconnected genes. This indicates that there is potential for further investigation into the role of RFX6 in tumors, drawing upon its known mechanism of action in diabetes. RFX6 may have utility as a biomarker for anticipating recurrence or metastasis, as well as for designing therapeutic strategies targeting RFX6.

Abbreviations

RFX6

Regulatory factor X6

RFX

Regulatory factor X

DBD

DNA-binding domain

MRS

Mitchell-Riley syndrome

ChIP-seq

Chromatin immunoprecipitation sequencing analysis

K_ATP channel

ATP-sensitive K⁺ channel

VDCCs

Voltage-gated Ca²⁺ channels

CN

Calmodulin phosphatase

NFATC2

Nuclear factor of activated T-cells 2

GIP

Gastric inhibitory peptide

NDM

Neonatal diabetes mellitus

PNDM

Permanent neonatal diabetes mellitus

TNDM

Transient neonatal diabetes mellitus

MFS

Martinez Frias syndrome

PGT

Primitive gut tube

MVT

Multi visual transplantation

GLP-1

Glucagon-like peptide-1

MODY

Maturity-onset diabetes of the young

DPP-4

Dipeptidyl peptidase-4

T2D

Type 2 diabetes mellitus

LUAD

Lung adenocarcinoma

ESCA

Esophageal carcinoma

PRAD

Prostate adenocarcinoma

LIHC

Liver hepatocellular carcinoma

GBM

Glioblastoma multiforme

BRCA

Breast invasive carcinoma

COAD

Colon adenocarcinoma

STAD

Stomach adenocarcinoma

LUSC

Lung squamous cell carcinoma

THCA

Thyroid carcinoma

OV

Ovarian serous cystadenocarcinoma

ACC

Adrenocortical carcinoma

PGAM1

Glycerate mutase 1

Author contributions

X.N. and J.Q. researched data and wrote the manuscript. X.N., J.Q. and Z.F. established the scope of the review. All authors made substantial contributions to the discussion and revision of the manuscript before submission. All authors read and approved the final manuscript.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 82174028), the Zhejiang Provincial Natural Science Foundation of China (No. LY22H280006) and Key Laboratory of Prevention, Diagnosis and Therapy of Upper Gastrointestinal Cancer of Zhejiang Province (2022E10021).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.