Deletion, translocation, amplification of chromosomes, and point mutations are often the reasons for the dysregulation of transcription factors (TF). Dysregulation of TF is a hallmark of cancer and is considered widespread in human malignant tumors(Sarnik et al. 2021; Paskeh et al. 2021; Gao et al. 2021). Forkhead box protein is a transcription factor that contains a wing helix DNA-binding domain (DBD). All members of the FOX family share this DBD but have different deactivation and inhibition domains(Bach et al. 2018). Specifically, the FOX family participates in the maintenance, progression, and metastasis of cancer at different levels of regulation. Notably, scholars have been involved in the study of transcription factors such as FOXA, FOXM1, FOXO, and FOXC in cancer. However, the detailed molecular mechanisms underlying the action of FOXD1, FOXD2, FOXD3, and FOXD4 in CRC remain unclear.
FOXD1, also known as FKHL8, FREAC4, and FREAC-4, is a protein-encoding gene. Research has shown that FOXD1 acts as a carcinogenic TF in cancers such as metastatic melanoma and renal clear cell sarcoma (Sun et al. 2021; Bond et al. 2021; Zhang et al. 2020; Li et al. 2019; Pan et al. 2018; Zhao et al. 2015). The overall survival rate of melanoma cells was reduced by high FOXD1 expression, the overexpression of which was positively associated with resistance to BRAFi or the combination of BRAFi and MEKi. Notably, FOXD1 knockdown restores sensitivity to BRAFi resistance(Sun et al. 2021). Similarly, FOXD1 and Gal-3 interact to form a positive feedback loop, which is a potential therapeutic target in lung cancer(Li et al. 2019). Moreover, FOXD1 activates the ERK1 /2 signaling pathway and promotes CRC development (Pan et al. 2018).
FOXD2, also known as FKHL17 or FREAC9, contains a distinct forkhead domain. As a protein-coding gene, FOXD2 was cloned from the human kidney and shares identical DNA-binding domains with FOXD1. A previous study showed that FOXD2 induces the expression of cAMP-dependent protein kinase RI α through its synergistic effect with protein kinase B, thereby increasing cAMP sensitivity(Johansson et al. 2003). Okabe et al. found that the expression level of FOXD2 mRNA is high in normal podocytes and that FOXD2 maintains podocyte integrity(Okabe et al. 2019). Notably, only three studies on FOXD2 in humans have been linked to cancer. Both meningiomas and CRC are associated with FOXD2 (Conesa-Zamora et al. 2015).
FOXD3 is an acidic, unstable, and hydrophilic protein that does not belong to the membrane or secretory proteins; notably, it has the greatest possibility of localization in the nucleus, affecting the regulation of many biological processes(Lam et al. 2013). Abnormal FOXD3 expression has been observed in melanoma, neuroblastoma, and other cancers(!!! INVALID CITATION !!! [38–46]). Thus, FOXD3 may exert antitumor effects. In addition, the Ras/Raf/MEK/ERK pathway was activated by silencing FOXD3(Li et al. 2017).
MAPK (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase play regulatory roles in tumors. Constitutive MAPK activation can trigger chemotherapy resistance in the cancer cells of multiple human malignancies. The RAS/RAF/MEK/ERK pathway is a key driver of human cancers(Gao et al. 2019; Nichols et al. 2018; Sanchez et al. 2019). Various cellular activities, such as cell proliferation and death, are regulated by this pathway. Inhibition of the MAPK pathway may be a potential research direction for early treatment. Furthermore, promoting the expression of FOXD3 is beneficial for the prognosis of patients with neuroblastoma(Li et al. 2013), and inhibiting FOXD3 expression is detrimental to the prognosis of breast cancer patients(Chu et al. 2014).
FOXD4, also known as FKHL9, FOXD4A, or FREAC5 is a transcription factor. As protein-coding genes, chromosome 9P deletion syndrome and cerebral palsy (Humphray et al. 2004; Chen et al. 2012) correlated with FOXD4. Interestingly, FOXD4 is associated with phenotypes such as obsessive-compulsive disorder and suicidal ideation(Minoretti et al. 2007) and is not conducive to CRC prognosis (Chen et al. 2018; Li et al. 2020).
In this study, we investigated the expression and clinicopathological characteristics of members of the FOXD subfamily. We also investigated their correlation with cell infiltration levels, related proteins, and their prognostic value, thereby providing a basis for further research. Here, we utilized IHC to validate the expression level of the FOXD subfamily by collecting CRC tissues from 20 patients. We found that FOXD1/2/3/4 expression was higher than that in adjacent tissues. These results were further confirmed by both WB and qPCR analyses.
In this study, we found that FOXD3 has a high overall diagnostic accuracy for CRC. The area under the ROC curve was 0.949. The results showed that FOXD1/3/4 were highly expressed, suggesting a poor prognosis. Therefore, FOXD1/3/4 may not only be prognostic biomarkers but also therapeutic targets. Furthermore, GSEA results showed that FOXD subfamily-related genes were mainly related to the focal adhesion and oxidative phosphorylation pathways.
The ECM–receptor interaction pathway contributes to cancer cell proliferation and invasion. ECM remodeling triggers the development of colitis-associated colon cancer. In this study, GSEA results showed that FOXD subfamily-related DEGs were positively correlated with the ECM receptor interaction pathway. However, the regulation of ECM reconstruction remains unclear. Notably, key genes in the FOXD subfamily were downregulated. Moreover, the hub genes of FOXD1 related DEGs were KRT5, KRT6A/6B/6C, KRT7, KRT14, KRT16, and KRT24.
There were 21 hub genes of FOXD2 related DEGs in PPI network including PPBP, CXCR1, and CXCL8, were identified in the PPI network. There were 13 hub genes of FOXD3-related DEGs, including PPBP. SPRR1A/1B, SPRR2E/2F, and SPRR3 were hub genes of FOXD4-related DEGs in the PPI network. Another important finding of the present study was that FOXD subfamily genes correlated with differential levels of immune infiltration in CRC. These results reveal that the underlying regulation of the FOXD subfamily affects ECM remodeling via the infiltration of immune cells.
In addition, we used Kaplan-Meier curves to analyze the overall survival of patients based on their FOXD1/2/3/4 levels. Our results revealed that FOXD1, FOXD3, and FOXD4 were prognostically significant. Subsequently, a nomogram prognostic model of the FOXD subfamily for CRC patients with CRC was developed. FOXD1, FOXD3, and FOXD4 have been confirmed to be independent prognostic factors for survival. The calibration curve revealed that the 3-year nomogram calibration curve had good predictive power.
However, the present study has some limitations. First, all clinical information was collected from public databases. We should collect more samples from our hospital and build a database for future research. Secondly, their differential expression was validated using IHC, WB, and qPCR. However, the underlying molecular mechanisms of FOXD subfamily genes in CRC have not been sufficiently investigated, especially in patients with metastases. Therefore, we plan to establish an animal model and siRNA oligo transfection, and then perform luciferase and Co-IP assays; further studies on the direct mechanism of FOXD subfamily genes in colorectal cancer are needed.
In conclusion, FOXD subfamily expression may be a good biomarker for predicting CRC prognosis and could serve as a novel therapeutic target. Further experimental validation is required to investigate the relationship between the FOXD subfamily and metastases in CRC.