The role of CSTF2 in cancer: from technology to clinical application

ABSTRACT

A protein called cleavage-stimulating factor subunit 2 (CSTF2, additionally called CSTF-64) binds RNA and is needed for the cleavage and polyadenylation of mRNA. CSTF2 is an important component subunit of the cleavage stimulating factor (CSTF), which is located on the X chromosome and encodes 557 amino acids. There is compelling evidence linking elevated CSTF2 expression to the pathological advancement of cancer and on its impact on the clinical aspects of the disease. The progression of cancers, including hepatocellular carcinoma, melanoma, prostate cancer, breast cancer, and pancreatic cancer, is correlated with the upregulation of CSTF2 expression. This review provides a fresh perspective on the investigation of the associations between CSTF2 and various malignancies and highlights current studies on the regulation of CSTF2. In particular, the mechanism of action and potential clinical applications of CSTF2 in cancer suggest that CSTF2 can serve as a new biomarker and individualized treatment target for a variety of cancer types.

1. Introduction

Cancer is a biologically diverse disease caused by seemingly abnormal changes in genetic material [1]. According to estimates for 2020, there were more than 10 million cancer-related deaths and 19.3 million new cancer cases worldwide (9.9 million of which were nonmelanoma skin cancers) [2]. Additionally, interactions involving the HLA (human leukocyte antigen) type, tumor microenvironment, immune cell repertoire, genetic polymorphisms, and microbiome, as well as those involving the tumor, host, contribute to the complex biology of malignancies [3]. Therefore, unrestricted proliferation, loss of contact inhibition, and distant metastasis are key factors in the metastasis and progression of cancer and the feature discrepancies between cancer cells and normal cells [4]. As a result, the dissociation of cancer cells from the tumor and spreading to distant sites continues to be a major contributor to the high cancer mortality rate. However, the low cure rates and poor prognosis are mostly due to the absence of generally stable, trustworthy, detectable biomarkers and potential therapeutic targets for cancer [5].

The long, Pro- and Gly-rich portion of CSTF2, the RNA-binding N-terminal RNP-type domain, and the extended alpha-helix pentapeptide repeat region [6,7]. The gene CSTF2 encodes a nuclear protein with an RNA binding domain of the ribonucleoprotein type that can control biological processes such as endoderm differentiation, cerebral development, and the generation of embryonic stem cells [6–8]. Furthermore, it has been discovered that CSTF2 controls the expression of genes in immune cells, such as T and B cells, suggesting a possible connection between CSTF2 and immune cell infiltration [9,10]. Recent studies have revealed that many malignant tumors have significant upregulation of CSTF2. CSTF2 may be involved in several processes that contribute to carcinogenesis by controlling the shortening of the 3’UTR (3’ untranslated region) of mRNA, which leads to the rapid proliferation of cancer cells, playing a role in the maintenance of stem cell self-renewal, immortalization of normal epithelial cells, early malignant transformation, etc [11,12]. For example, it has been shown that CSTF2 is overexpressed in non-small cell lung cancer (NSCLC) and can promote cancer progression [13].

However, CSTF2 has not been extensively investigated for its pancancer regulatory role. It has been suggested that CSTF2 plays an important role in the activation of some oncogenes by altering the length of the 3’UTR of these oncogenes. As a key nuclear regulator, CSTF2 plays an unknown role in many cancers. CSTF2 plays a regulatory role in a variety of malignancies, as discussed in this review. On this basis, we also explored potential clinical applications for CSTF2 in the detection, management, and cancer prognosis (Tables 1 and 2).

Table 1.

The roles of CSTF2 in various cancers.

CSTF	Cancer type	Cell/Tissue	Expression change (the proportion of cases with amplified CSTF2)	Evidence supported function in cancer carcinogenesis
CSTF2	NSCLC	NCI-H1781,NCI-H1373,LC319,A549,PC-14,SK-MES-1,NCI-H520,NCI-H1703,NCI-H2170,LU61,SBC-3,SBC-5,DMS114,DMS273, LX1	+a	Therapeutic target /prognosis marker13
CSTF2	UCB	J82, T24, 5637	+a	Therapeutic target /prognosis marker32
CSTF2	HCC	Huh7	+a	Therapeutic target /prognosis mark86
CSTF2	TNBC	MCF10A and MDA-MB-468	+a	Therapeutic target35
CSTF2	PDAC	PANC-1 and SW1990	+a	Therapeutic target /prognosis marker66
CSTF2	ESCC	KYSE30 and KYSE450	+a	Therapeutic target33

a “+” indicates an increase in expression.

Table 2.

The molecular complex or signaling pathway associated with CSTF2 in cancer.

CSTF	Cancer type	Target or associated molecular/complex	Pathway
CSTF2	NSCLC	/	TGF-beta signaling pathway and Heterotrimeric G-protein signaling pathway-Gq alpha[13]
CSTF2	UCB	RAC1[14]	/
CSTF2	HCC	/	PI3K/AKT/mTOR pathway[15]
CSTF2	TNBC	EGF [16]	/
CSTF2	PDAC	TRAF1 and TRAF[17]	/
CSTF2	ESCC	BID[18]	/

2. Overview of cancer

Cancer is widely believed to be a genetic disease characterized by mutations that drive its progression [19]. Cancer is a leading cause of death worldwide, but the burden is unequally distributed [20]. With a growing and aging global population, cancer is a major cause of premature death and has reduced life expectancy in many countries. In 2020, it was expected that breast cancer would overtake lung cancer as the most commonly diagnosed cancer worldwide [21]. Cancers may form when tissue cells proliferate abnormally in response to physical, chemical, biological, or other carcinogenic factors [5]. Usually, cancer is caused by mutations, epigenetic changes, or alterations in key genes that regulate cell reproduction, division, metabolism, and growth. According to the effect they produce when mutated, these genes are classified as oncogenes and tumor suppressors [22,23]. Cancer cells undergo significant changes in their growth and metabolism under the influence of abnormal gene expression. It is common for cancer cells to have altered metabolic states that affect the signaling pathways for glucose, glutamine, and fatty acids. As a result of these changes, cancer cells produce ATP (adenosine triphosphate) and metabolic intermediates that sustain their proliferative state [19]. For example, anaerobic respiration is the main mode of intracellular respiration; it wastes a large number of nutrients and leads to cachexia in cancer patients at the end of their lives [24]. Early detection, supportive treatment, and precise treatment have all played a major role in improving cancer survival rates in recent decades [24]. To enhance the early identification, precise therapy, and prognostic assessment of cancer, the search for diagnostic biomarkers and targets for therapeutic intervention is of the utmost importance.

3. Overview of CSTF2

CSTF2 regulates mRNA polyadenylation sites through its RNA binding function. As a result of strong and specific cross-linking of CSTF2 with poly (A)-containing RNA, the protein was first identified as part of the complex that processes pre-mRNA at its 3” end [25,26]. CSTF2 is a member of the CSTF family of cancer-related genes that can affect many biological processes [6]. Through the 64-kDa subunit, CSTF is linked to the downstream component of the 3” process [27]. CSTF2 can encode an RNA-binding protein with an N-terminal RNA-binding structural domain that is similar to that found in ribonucleoproteins (Figure 1). Together with two other cleavage-stimulating factors, CSTF2 has been shown to contribute to the polyadenylation of mRNA in HeLa cells [28]. CSTF2 is implicated in the regulation of selective polyadenylation of many genes, and changes in CSTF2 expression levels under specific conditions are associated with the regulation of selective polyadenylation in immune cells. CSTF2 recognizes the highly conserved AAUAAA hexanucleotide downstream of the point of cleavage and other polyadenylation sites with GU-rich 3’UTRs [29]. Coexpressed genes collaborate to modulate specific pathophysiological processes and thus possess similar functional roles. Thus, the analysis of genes coexpressed with CSTF2 is an outstanding approach to comprehend CSTF2 oncogenic mechanisms. NONO, CENPI, and PHF6 were found to be the top three genes that are positively correlated with CSTF2 [30]. These genes have been implicated in the development and progression of cancer. NONO, a protein that binds to RNA and DNA, promotes tumorigenesis by driving oncogenic transcriptional programs [31]. CENPI, belonging to the centromere protein family, is crucial for precise chromosome segregation [32]. CENPI is upregulated in multiple cancers and serves as an oncogene to promote tumor cell proliferation, migration and invasion [33,34]. As a protein that binds to chromatin, PHF6 is upregulated in cancer and shows a positive correlation with lymph node metastasis [35,36]. These findings suggest that CSTF2 and its corresponding genes hold potential as reliable prognostic biomarkers for cancer. In urothelial carcinoma of the bladder (UCB), CSTF2 promotes the proliferation, migration and invasion of cancer cells [14]. By limiting competitive endogenous RNA cross-talk and suppressing the expression of the tumor suppressor gene ZFP36L2, the shorter BID (a proapoptotic member of the B-cell lymphoma-2 family) 3’UTR produced by CSTF2 accelerates the development of esophageal squamous cell carcinoma (zinc finger protein 36, C3H type-like 2) [18]. In lung cancer, CSTF2 may control cancer-related genes by acting as an oncogene [37]. Therefore, CSTF2 may represent a new generation of therapeutic targets and cancer biomarkers.

Figure 1.

Domains of human CSTF2.

4. Functional role of CSTF2 in various cancer types

CSTF2 is a key player in the development and clinical features of many cancers, according to a growing body of research. It has been reported that some types of malignancies have increased CSTF2 expression, including NSCLC [13], UCB [14], hepatocellular carcinoma (HCC) [30], triple-negative breast cancer (TNBC) [16], pancreatic ductal adenocarcinoma (PDAC) [38], and esophageal squamous cell carcinoma (ESCC) [18], indicating a significant contribution to the pathological development of cancer. We also analyzed CSTF2 transcriptional levels in different cancers via TIMER (Tumor Immune Estimation Resource, https://cistrome.shinyapps.io/timer/) (Figure 2). Therefore, this paper summarizes the role of CSTF2 in six types of cancer and summarizes its specific mechanism of action.

Figure 2.

CSTF2 transcript levels in various cancers (analyzed in the TIMER database).

4.1. Lung cancer

Lung cancer is the second most common disease in the world, with a very poor overall survival rate of approximately 18% at 5 years [39]. NSCLC accounts for 80% of deaths from lung cancer, which is the most common type of cancer worldwide [40]. Despite the fact that there are other subtypes of lung cancer, historically, small cell lung cancer and NSCLC have been considered to be the two main types [41]. In recent years, significant advancements have been achieved in the clinical diagnosis and treatment of NSCLC. Although a number of novel cytotoxic agents have been developed, these regimens have shown only moderate survival benefits compared to traditional platinum-based regimens. Therefore, there is a research push to explore new biomarkers to improve the efficacy of combination therapy. Yataro Daigo et al. [13] used cDNA microarrays consisting of 27,648 genes or expressed sequence tags to analyze genome-wide gene expression profiles of 120 lung cancers and found that CSTF2 was overexpressed. Among the lung cancer cell lines analyzed, H460 cells showed the highest expression of CSTF2. Further analysis of the CSTF2 expression pattern in lung cancer samples was performed using The Cancer Genome Atlas, where CSTF2 expression in cancer cells was almost twofold higher than that in normal lung cells [37]. CSTF2 siRNA was transfected into A549 and LC319 cells (lung cancer cell lines) overexpressing CSTF2, and the expression level of CSTF2 was significantly reduced, along with the cell viability rate and colony number [42–45]. It is common for 3’UTRs to undergo alternative polyadenylation (APA), and CSTF2 may act as an oncogene by controlling the length of 3’UTRs on cancer-associated genes in NSCLC. The length of the 3’UTR is crucial in controlling mRNA stability, localization, and protein translation efficiency, as the 3’UTR of an mRNA often contains a large number of binding sites for regulatory RNA-binding proteins and miRNAs [46], which is detrimental to NSCLC development. In summary, these results not only highlight the importance of CSTF2 in regulating the mRNA 3’UTR length of genes associated with cancer cells but also suggest the possibility that CSTF2 or members of its protein family may play a role as oncogenes in promoting NSCLC carcinogenesis.

4.2. Bladder cancer

Bladder cancer, the most common malignant cancer of the urinary tract, is estimated to account for 573,000 new cases and 212,000 related deaths worldwide each year². UCB is the most common histological form of bladder cancer, accounting for 90% of all confirmed cases of bladder cancer [39]. To increase survival, systemic therapy is necessary for patients with locally advanced or metastatic urothelial bladder cancer. Although platinum-based chemotherapy remains the standard of care for metastatic urothelial cancer, it has limited long-term efficacy and tolerability in the elderly [47]. In two UCB tissue cohorts, Xie Dan et al. [14] investigated the relationships between the expression of six significant cleavage/polyadenylation factors and the use of the RAC1 (Rac family small GTPase 1) short-3’UTR. According to the results, RAC1 short 3’UTR usage in UCBs correlated positively with CSTF2 expression levels. An oncogene called RAC1 is critical for the malignant character of many different types of human cancer [48,49]. Therefore, CSTF2 supports the invasive and oncogenic properties of the RAC1 short 3’UTR isoform in UCB cells. They found that levels of the RAC1 short 3’UTR isoform in UCB cells were significantly lower in the CSTF2 knockdown group than in the control group and that this had a significant inhibitory effect on colony formation, migration and invasion. However, these effects were significantly reversed by the forced overexpression of the short-3’UTR variant of RAC1 in CSTF2-silenced UCB cells. Moreover, researchers have also found that overexpression of CSTF2 interacts with the GUKKU motif downstream of RAC1 mRNA to prevent AFF1- and AFF4-mediated transcription elongation, interferes with the transcription of other genes, induces shortening of the 3’UTR gene of RAC1 mRNA in UCB cells, and plays a significant oncogenic role in the pathogenesis of UCB [14,50,51] (Figure 3). By evading miRNA-targeted regulation, the short 3’UTR isoform of RAC1 significantly increased RAC1 expression and contributed to the pathogenesis of UCB. An essential cleavage/polyadenylation factor called CSTF2 caused delayed transcriptional elongation at RAC1 to shorten the 3’UTR of RAC1 in UCB. Importantly, our findings may provide a new therapeutic target, CSTF2, which could expand human therapeutic options for UCB.

Figure 3.

Schematic diagram depicting a proposed model for the role and a major mechanism of RAC1 3’UTR shortening induced by CSTF2 in UCB.

4.3. Hepatocellular carcinoma

Among primary liver cancers, HCC is the most common pathological type and is a common malignant tumor of the gastrointestinal system [52]. The 5-year survival rate of patients with HCC in China has been reported to be only 14%, with a recurrence rate of up to 70% at 5 years after surgery [53–55]. Xuan Zhu et al. [30] used bioinformatics to investigate the expression of the CSTF2 gene in HCC. TCGA analysis showed that the intensity of the CSTF2 gene was abnormally increased in HCC compared with normal tissue. In Huh7 and HCCLM3 cells (liver cancer cells), CSTF2 overexpression and knockdown were successfully performed. The impact of differential CSTF2 expression on the characteristics of transfected Huh7 and HCCLM3 cells was subsequently evaluated, and it was found that CSTF2 overexpression not only significantly inhibited Huh7 apoptosis but also significantly enhanced Huh7 cell invasion and migration. CSTF2 overexpression also enhanced colony formation and cell proliferation. On the other hand, knocking down CSTF2 in HCCLM3 cells had the opposite effect. These results showed that CSTF2 was highly expressed in HCC cells, which promoted the growth of cancerous phenotypes. In summary, abnormally elevated CSTF2 gene expression in HCC correlates with the malignant progression-related characteristics of patients and is expected to be an independent prognostic factor and potential therapeutic target for patients with HCC.

4.4. Triple-negative breast cancer

TNBC is immunobiologically characterized by estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene Her-2 proto-oncogene negativity and is characterized by aggressiveness, high proliferation index, early time to recurrence and high mortality [56]. TNBC typically presents in younger women and has a higher mortality rate of 40% in the first five years after diagnosis when it is at an advanced stage [57,58]. The median survival time is 13.3 months, and approximately 45% of patients with advanced-stage TNBC will develop distant metastases to the brain and/or visceral organs [59]. Despite the introduction of novel biologic and targeted medicines, cytotoxic chemotherapy continues to be the cornerstone of treatment for TNBC [60]. Epidermal growth factor (EGF), when administered to the human mammary epithelial cell line MCF10A and the breast cancer cell line MDA-MB-468, caused TNBC cell proliferation as a result of the combined action of CSTF2 and EGF. The CSTF2 gene was highly expressed in EGF-treated MCF10A and MDA-MB-468 cells compared to untreated cells, and cell proliferation was accelerated. CSTF2, a critical factor in the selection of proximal polyadenylation sites in EGFR (epidermal growth factor receptor) positive TNBC cell lines. The 3’UTR lengths of multiple transcripts are expected to change simultaneously as a result of EGF responsive APA. EGF increases CSTF2 levels, and poly(A) site selection may be influenced by increased expression of APA machinery proteins [16]. In the regulation of gene expression, APA regulates gene expression by shortening the 3’UTR of the proliferation signal and slowing microRNA-mediated repression, and CSTF2, a key factor controlling 3’UTR shortening, was found to be upregulated by EGF [61]. Taken together, these results suggest that CSTF2 has an overall effect on transcription as one of the APA machineries, thereby altering the growth characteristics of cells, and is expected to be a new target for TNBC therapy.

4.5. Esophageal squamous cell carcinoma

Esophageal cancer is the ninth most commonly diagnosed cancer worldwide and ranks sixth in terms of the number of cancer-related deaths [2]. Approximately 90% of esophageal cancer cases worldwide are ESCC, the most common histological subtype of the disease [62]. Multimodal therapy, including surgery, radiotherapy, and chemotherapy, is currently the mainstay treatment modality for ESCC [63]. Therefore, there is a need to improve the prognosis of ESCC patients, which requires the rapid identification of novel biomarkers and molecular targets. Eric J. Wagner et al. [18] used RNA-seq of APA to dynamically analyze the 3’UTR APA profile in ESCC and found recurrent 3’UTR shortening in BID mRNA and a significant increase in BID mRNA expression level in ESCC, further revealing that the length of the 3’UTR of BID mRNA is potentially regulated by CSTF2. CSTF2 was silenced in the esophageal cancer cell lines KYSE30 and KYSE450, and the usage of BID poly(A) sites was detected by qRT‒PCR, which showed that silencing the CSTF2 gene resulted in a lengthening of the BID 3’UTR and a significant decrease in BID expression. However, the BID 3’UTR was shortened, and BID expression was elevated as a result of CSTF2 overexpression. In contrast, overexpression of CSTF2 shortened the BID 3’UTR and increased BID expression. Collectively, these findings imply that CSTF2 expression affects the 3’UTR length of BID mRNA. CSTF2 is an early regulator of the 3’UTR in BID mRNA, and shortening of the 3’UTR in BID mRNA promotes ESCC cell proliferation by interfering with competing endogenous RNA (ceRNA), resulting in the downregulation of the tumor suppressor gene ZFP36L2. ZFP36L2, an RNA-binding protein that promotes deadenylation and degradation of mRNA, binds to AU (nucleobase)-rich regions in the 3’UTRs of target genes [64]. This suggests that CSTF2 expression-induced BID 3’UTR shortening can enhance tumorigenesis by disrupting the ceRNA network with ZFP36L2 (Figure 4). Therefore, CSTF2 regulates the recurrent shortening of the BID 3’UTR, which is a driver of ESCC progression and may be a novel molecular target.

Figure 4.

In cancer cells, upregulation of CSTF2 shortened the 3’UTR of BID, and released miR-124-3p and miR-506-3p to repress its ceRNA partner ZFP36L2 in trans.

4.6. Pancreatic ductal adenocarcinoma

PDAC is one of the deadliest and most invasive types of malignant tumors. With a less than a 10% chance of survival, PDAC patients have a very poor prognosis [39]. The typical survival period for PDAC patients is less than one year, and the majority of patients have advanced or metastatic disease when they are first diagnosed. Standard treatment strategies, including radiation and chemotherapy, only slightly extend the overall survival time of patients [65]. Immune checkpoint blockade (ICB) therapy has recently shown encouraging clinical results in a variety of cancers; however, PDAC is often regarded as a less immunogenic malignancy [66,67]. A single-sample gene enrichment analysis was performed by Huafang Su et al. [17]. Examination of 119 immune gene signatures identified CSTF2 as a risk factor associated with immune indices and revealed that CSTF2 may contribute to the diagnosis of PDAC and may be a marker for therapeutic targets. Zhixiang Zuo et al. [38] explored the effect of CSTF2 on the malignant phenotype of PDAC cells (PANC-1 and SW1990). In vitro experiments showed that knockdown of CSTF2 significantly inhibited the proliferation, colony formation, cell cycle, migration, and invasive ability of PDAC cells. Using a mouse subcutaneous xenograft model, we also found that overexpression of CSTF2 significantly enhanced the growth rate of PDAC tumors, but silencing CSTF2 significantly inhibited growth. Furthermore, forced expression of CSTF2 promoted the lung metastasis of PDAC cells, whereas knockdown of CSTF2 induced the opposite effect. Moreover, malignant phenotypic inhibition induced by CSTF2 knockdown could be rescued by forced expression of CSTF2. Collectively, these findings suggest that CSTF2 is involved in the development and progression of PDAC and may prove to be a useful prognostic indicator connected to immunological indices. By regulating the shortening of the mRNA 3’UTR, CSTF2, a central member of the CSTF complex, may be involved in a number of processes that promote carcinogenesis. Therefore, the trend of mRNA 3’UTR shortening may be related to the overexpression of CSTF2. Along with the above-stated strategies, a few upstream genes and associated signaling pathways may also contribute to CSTF2 expression. Overall, the significance of CSTF2 in different malignancies is being continually revealed, and this is a field that will require consideration in the future.

5. Regulatory mechanism of CSTF2 in cancer

It is well known that complicated signaling pathways are involved in the development, spread, and other characteristics of cancer and that these processes contribute to the dismal prognosis of the condition. CSTF2 participates in the Wnt-catenin signaling pathway, glycolysis, Notch signaling pathway, and P53 signaling pathway. All of these pathways are involved in cancer development and progression [68–72]. Therefore, this section highlights how CSTF2 is involved in tumorigenesis and progression through two signaling pathways: the phosphatidylinositol 3’-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway [15] and the EGF signaling pathway, which regulates the shortening of the 3’UTR [16].

Numerous biological processes, including cell survival, proliferation, differentiation, metastasis, apoptosis, metabolism, and angiogenesis, have been shown to involve the PI3K/AKT/mTOR signaling pathway [73,74]. An earlier investigation demonstrated that PI3K/AKT signaling pathway activation increased the metastasis and invasion of squamous cell carcinoma cells [75]. Another investigation demonstrated that deactivating the PI3K/AKT/mTOR signaling pathway allowed anemoside B4 to exert its anticancer effects in HCC [76]. CSTF2, a part of the CSTF complex, has been found to be an oncogene in a variety of cancers. The expression of CSTF2 is increased in various cancers, such as colon adenocarcinoma, liver cancer, uterine corpus endometrioid carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, and lung adenocarcinoma [77–80]. As evidenced by the significantly elevated P-AKT/AKT, P-PI3K/PI3K and p-mTOR/mTOR ratios, CSTF2 overexpression supported the development of malignant phenotypes in HCC cells by activating the PI3K/AKT/mTOR signaling pathway. In contrast, CSTF2 knockdown reduced these ratios, indicating inactivation of this pathway. These findings suggest that CSTF2 can activate the PI3K/AKT/mTOR signaling pathway to stimulate HCC cell proliferation, migration, and invasion while suppressing apoptosis.

The EGF signaling pathway is needed to promote epithelial cell formation and self-renewal, which is necessary to maintain normal epithelial cells [81]. Hexameric procyanidins reduce the development of colorectal cancer cells by regulating the EGF signaling pathway both redoxly and nonredoxly [82]. When EGFR interacts with its natural ligand EGF, it increases tyrosine phosphorylation and receptor dimerization with other family members, which promotes unchecked proliferation [83]. The autophosphorylation of the EGF receptor and downstream signaling proteins, including Akt kinase and extracellular signal-regulated kinase 1/2, is inhibited by EGFA inhibitors, which also cause cancer cell death [84]. CSTF2 expression levels increase in response to EGF, and increased APA machinery protein expression influences poly(A) site selection decisions while altering the 3’UTR length of hundreds of transcripts and promoting cancer progression [85]. The EGFR signaling pathway was specifically inhibited pharmacologically by AG1478 (a selective tyrosine kinase inhibitor of EGFR), which also reduced pEGFR levels and stopped the increase in CSTF2 protein and mRNA levels. Notably, treatment with AG1478 led to a further reduction in CSTF2 levels. CSTF2 is a component of the APA machinery, and as such, it affects transcripts in general, which in turn affects cell growth. EGF causes CSTF2, a key regulator of 3’UTR shortening, to be upregulated.

In conclusion, CSTF2 has a substantial impact on the development and healing of tumor cells because it regulates a variety of cellular signaling pathways, which are critical for the initiation, progression, and clinical manifestations of a wide range of cancers. This demonstrates that CSTF2 could be used as a beneficial treatment target and a critical predictor of cancer prognosis and that the gene might serve as a novel biomarker and specific treatment target for certain malignancies.

6. Clinical applications

Cancer is difficult to detect in its early stages without overt clinical symptoms; this contributes to the low survival rate of cancer patients, as patients diagnosed in late stages are more likely to miss the best window for treatment. Therefore, the identification of sensitive cancer biomarkers that can detect molecular variations in the early stages of certain cancer types would aid in early diagnosis. Consequently, this article provides a summary of research into the use of CSTF2 as a marker for cancer diagnosis and prognosis and metastasis prediction.

The diagnostic significance of CSTF2 has been demonstrated in several studies in recent years. Yataro Daigo et al. [13] reported that most of the lung cancer samples they examined had overexpressed CSTF2 transcripts (3-fold or greater), whereas none of the 29 normal organs other than the testis had high CSTF2 expression. The database was used by Huiming Xu et al. [86] to further investigate the relationship between the degree of clinical malignant HCC and high CSTF2 expression. The ROC (receiver operating characteristic) curve for CSTF2 was created using data from TCGA to evaluate the diagnostic usefulness of CSTF2 in HCC. The findings indicated that CSTF2 had a high degree of discriminatory power in the diagnosis of HCC patients, with an AUC (area under curve) of 0.938 (95% CI 0.913–0.964), and that higher CSTF2 expression was associated with more advanced clinical stage in HCC patients (P < 0.05), indicating an association between CSTF2 expression and clinicopathological characteristics of HCC patients. Through using TCGA RNA-seq data analysis, Dan Xie et al. [14] discovered that CSTF2 mRNA expression was markedly increased in UCB tissues (P < 0.05). Further statistical analysis showed that UCB patients with high expression of the RAC1 short 3’UTR isoform had worse disease-free survival (DFS, P < 0.05), greater tumor size and advanced T and N status. In summary, these results statistically demonstrate the potential of CSTF2 as a diagnostic biomarker.

In addition to its diagnostic value, the prognostic value of CSTF2 is very strong. For example, in lung cancer studies, correlation analysis of CSTF2 expression levels on tissue arrays and various clinicopathological variables revealed that patients with NSCLC who underwent primary tumor excision had a poor outcome (P = 0.0079) and had significant CSTF2 expression [13,87]. The clinical data of HCC patients from TCGA-HCC were examined by Wang Zhang et al. According to the data, there was a significant relationship between CSTF2 expression and sex (P = 0.037), AJCC (American Joint Committee on Cancer) stage (P = 0.003), and survival status (P = 0.008). The expression of CSTF2 did not significantly correlate with other clinicopathological factors, such as age (P = 0.59) or histologic grade (P = 0.099). In addition, Cox proportional hazards analysis based on the TCGA database showed that HCC patients with high CSTF2 expression had worse overall survival (OS) (P < 0.05) and relapse-free survival (RFS) than those with low expression (P < 0.05). Furthermore, the study revealed that CSTF2 expression served as an independent prognostic factor for predicting overall survival in the multivariate Cox analysis [hazard ratio (HR): 1.671, 95% confidence interval (CI): 1.023 to 2.728, P = 0.040]. Together, these findings suggest a link between advanced clinical stage and a poor prognosis for patients with increased expression of CSTF2.

According to the aforementioned studies, CSTF2 may be crucial for cancer diagnosis, prognosis, and treatment. To clarify the clinical significance of CSTF2 in cancer, more thorough investigation and additional confirmation studies are needed because the precise underlying mechanism is still unclear.

7. Future expectations

Through substantial research, the mechanisms underlying the function of CSTF2 in cancer are becoming clearer. We discovered that elevated levels of CSTF2 drive the oncogenic process in NSCLC, UCB, HCC, TNBC, PDAC, and ESCC malignancies and subsequently play a crucial regulatory function in invasion and metastasis as well as prognosis. Furthermore, CSTF2 has been shown to be a probable oncogene in some malignancies and is closely associated with cancer development through specific signaling pathways. Thus, it is anticipated that CSTF2 will be a novel treatment target for cancer. In the future, CSTF2 targeting combined with cutting-edge technology may facilitate discoveries in the life sciences and medical field.

In recent years, there have been notable advancements in cancer treatment. However, the accumulation of toxicity and harmful side effects drastically lowers the quality of life for cancer patients. We are aware that a number of alterations in the genome and signaling pathways, primarily leading to the activation or inactivation of oncogenes, are the primary causes of cancer. Therefore, researchers are focusing on the activation of oncogenes and the signaling pathways of cancer to improve cancer treatment outcomes and prognosis. The use of CRISPR/Cas9 gene editing technology in cancer immunotherapy research has been extensively studied [88,89]. CRISPR/Cas9, an RNA-guided genome editing tool, was first tested in mammals in 2013. CRISPR/Cas9 provides several advantages over conventional genome editing techniques, including ease of use, safety, high efficiency (approximately five times that of conventional technology), and multiplexing (able to edit multiple genes simultaneously). Additionally, several scientists have used the CRISPR/Cas9-mediated CPSF3 (cleavage and polyadenylation specificity factor subunit 3) gene in combination with AN3661 to reduce human resistance to antimalarials [90]. CPSF3, one of the major parts of the CPSF complex, is an RNA-binding protein [91,92]. Additionally, CPSF and CSTF play important roles in the processing of the 3’ end of mRNA. We can easily imagine that using CRISPR/Cas9 to inhibit oncogenes such as the CSTF2 gene could be a viable cancer therapy. In addition, the use of catalytically inactivated dCas9 as a site-specific DNA-binding domain and fusion with epigenetic modifiers to form a complex that enters the target site under the control of sgRNAs and performs epigenetic modification, a process known as epigenome editing, is another potential direction for the use of CRISPR‒Cas9 to treat cancer [93,94]. Therefore, as an epigenetic modulator and matching gene expression product, the CSTF2 protein may also be suitable for epigenome editing therapy. Together, these findings suggest that CSTF2 oncogene silencing and protein fusion may be used in the future to treat cancer using CRISPR/Cas9 technology.

Through a number of mechanisms, including the prevention of cell division, metastasis, and angiogenesis, the induction of apoptosis, and the reversal of multidrug resistance, molecular targeted therapies produce anticancer effects [95]. Several molecular targeted therapeutic drugs, either alone or in combination with chemotherapeutic treatments, promote host antitumor immunity by enhancing CD8 T-cell recruitment and natural killer cell cytotoxicity, suppressing immunosuppressive myeloid cells, and triggering immunogenic cell death [96]. The S. digitata cDNA library was created to find new therapeutic targets for the treatment of HLF (human lymphatic filariasis), and a 549 bp cDNA fragment was sequenced and analyzed in silico. The results show that the shortest RNA recognition motif of 249 bp from the cDNA encodes a polypeptide of 82 amino acids and shows 54% amino acid identity with the RRM domain of human CSTF2. The RNA recognition motif (RRM) of CSTF2 was used as a template for the homology modeling process, which produced a protein with a structure that follows the traditional RRM topology. The structure of the protein model was confirmed using superposition methods and Ramachandran plot analysis. Automated molecular docking of RRM of CSTF2 with the same three types of RNA octamers results in comparable results as obtained for the structure of the protein, revealing that the structure of the protein has significant functional similarity with human CSTF2 [97]. Overall, we naturally suggest the functional analysis of RNA binding proteins of CSTF2 and their evaluation as new drug targets against cancer.

In conclusion, CSTF2 is certainly a promising area of study that is not only at the forefront of life science and medical research but has also been linked to a number of groundbreaking discoveries in the field. In addition, CSTF2 controls a number of signaling pathways that are active in cancer cells. CSTF2 promotes cancer cell proliferation and division and is highly expressed in NSCLC, UCB, HCC, TNBC, PDAC, ESCC, and CCA. According to the aforementioned findings, CSTF2, from gene to protein, is not only implicated in several pathways of cancer growth but also influences its clinical manifestations, with potential translational uses. We think that CSTF2 will undoubtedly offer fresh approaches to comprehending and treating malignancies.

8. Expert opinion

The following characteristics of cancer cells are their main distinguishing characteristics: self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, infinite capacity for replication, sustained angiogenesis, reprogramming of energy metabolism, avoidance of immune destruction, tissue invasion, and metastasis [98]. Another crucial feature of cancer cells is their ability to attract a variety of healthy cells that support cancer growth, including fibroblasts (the most common cell type), pericytes, endothelial cells, mesenchymal stem cells, macrophages, and lymphocytes [98]. As a promoter of tumorigenesis and metastasis, CSTF2 is an interesting therapeutic target and prognostic marker for cancer. Therefore, CSTF2 inhibition may have additional relevance in the context of targeted cancer therapy.

Could CSTF2 target cancer therapy and improve the prognosis of cancer patients? No CSTF2-specific inhibitors currently exist. However, developing drug inhibitors that target other RNA binding proteins (RBPs) has been an area of active research, and multiple successful efforts have provided proof of feasibility. Additionally, CSTF2 inhibitors have been discovered in chemical screens and created through rational drug design [99]. Notably, an RBP inhibitor discovered through a chemical screen has demonstrated action against cancer both in vitro and in vivo. The number of inhibitors that target different RBPs is also continuing to expand [100].

In summary, epigenetic processes have become powerful candidate suppressors, silencers and inducers of oncogenic pathways in cancer. Epigenetic alterations are frequently observed in cancer and, in the case of hypomutated tumors such as those of infancy, provide an alternative method for the genesis and development of malignant phenotypes. Encouragingly, several epigenetic pathways can be targeted therapeutically. A growing number of different malignancies have shown that CSTF2 promotes a variety of cancer characteristics. Given the many links between CSTF2 and metastasis and other features important for disease development, CSTF2 represents an exciting new therapeutic target with the potential to affect outcomes in both common adult cancers and pediatric tumors such as neuroblastoma and glioblastoma.

Funding Statement

This study was supported by the 512 Talent Cultivation Program of Bengbu Medical College [by51201313], Young Scientist Fund of Bengbu Medical College [2021byyfyyq02] Anhui Provincial Natural Science Foundation [KJ2021A0815], Anhui Medical University Clinical Foundation (2020xkj208), Anhui Biochemical Engineering Centre Research Platform Project [2023SYKFZ06].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

JD and YS conceived the presented study and drafted the manuscript. YL designed the figures and tables. YX reviewed the manuscript. DY modified the figures and tables. XW revised the grammar. HS, HZ and HL conceived the presented study and reviewed the draft. JD submitted the document for publication. All authors agreed on the final version.

Abbreviation

CSTF2/CSTF-64: cleavage stimulation factor subunit 2; CSTF: cleavage stimulating factor; HLA: human leucocyte antigen; 3’UTR: 3’untranslated region; NSCLC: non-small cell lung cancer; ATP: adenosine triphosphate; UCB: urothelial carcinoma of the bladder; BID: a pro-apoptotic member of the B-cell lymphoma-2 family; ZFP36L2: zinc finger protein 36, C3H type-like 2; HCC: hepatocellular carcinoma; TNBC: triple-negative breast cancer; PDAC: pancreatic ductal adenocarcinoma; ESCC: esophageal squamous cell carcinoma; TIMER: Tumor Immune Estimation Resource; APA: alternative polyadenylation; RAC1: Rac family small GTPase 1; ER: estrogen receptor; PR: progesterone receptor; EGF: epidermal growth factor; EGFR: epidermal growth factor receptors; ICB: immune checkpoint blockade; PI3K/AKT/mTOR: phosphatidylinositol 3’-kinase/protein kinase B/mammalian target of rapamycin; CPSF3: cleavage and polyadenylation specificity factor subunit 3; HLF: human lymphatic filariasis; RRM: RNA recognition motif; RBP: RNA binding proteins;