Alternative splicing of the androgen receptor is one of the most studied events for PCa. This nuclear receptor for male sex hormones is a multidomain protein that possess the ability to dimerize when binding to an androgen molecule and the activation of this route starts different mechanisms of cell development and proliferation (42).

The gene coding for the AR consists of 8 exons, where exons 5 to 8 encode the ligand-binding domain (LBD). Interestingly, in prostatic cancer cells the expression of this gene is modified and an aberrant alternative splicing occurs ( Figure 1A ), generating two truncated isoforms of the receptor, named AR-V7 and AR-V9 (43). These isoforms completely lack the LBD domain and therefore they are not able to bind to androgens, but they are constitutively active, so that they cannot be negatively regulated and will be constantly generating proliferation, conferring resistance and protection against hormonal treatments (15–17).

The expression of these aberrant isoforms derived from a deregulated alternative splicing event is not directly associated with a mutation in the gene for the androgen receptor, however it has been pointed out that the level of expression of these isoforms is related to an increase in the number of copies within the locus containing the gene in the cell. Moreover, it has been depicted that a deregulation and overexpression of the SRSF1 splicing factor correlates with the appearance of aberrant isoforms (18).

The BCL-X gene (or BCL2L1) regulates apoptosis and therefore has an extremely important role in cancer (19, 20). This gene is constituted by 3 exons with the ability to generate 2 isoforms due to the selection of two alternative 5’ splice sites in exon 2, resulting in isoforms BCL-X_L (B-Cell Lymphoma xtra large) and BCL-X_S (B-Cell Lymphoma xtra small). Both isoforms have opposite functions where BCL-X_S is proapoptotic being part of the core apoptosome, while on the contrary BCL-X_L is antiapoptotic by antagonizing proapoptotic functions of other molecules belonging to the BCL-2 family ( Figure 1B ).

The regulation of the expression of these isoforms is determined by the regulation of the cell cycle and with this, the activation of different phosphatases. At the level of splicing regulation, it is known that the expression of BCL-X_S is determined by hnRNP H and F, SAM68 and RBM25 (44–46). On the other hand, the expression of BCL-X_L is regulated by SR proteins such as SRSF1, SRSF9 and SAP155 (45–48) that must be phosphorylated and this modification usually occurs during the initiation of cell replication (22). The disruption of the delicate balance between BCL-X_L and BCL-X_S isoforms has not been related to a specific mutation; however, it is believed that it has to do with the appearance of epigenetic changes that affect the cell cycle, but further evidence is needed to fully demonstrate this observation.

In the context of PCa, it has been determined that prostatic cell lines expressing BCL-X_L show a fairly high resistance to apoptosis generating resistance to various treatments from chemotherapy to radiotherapy (21). As an alternative, phosphorylase inhibitor drugs have been implemented for cancer treatment, although this could generate negative effects in other healthy tissues.

The CCND1 gene consists of 5 exons and encodes the cyclin D1 protein which is a proto-oncogene given its ability to activate cyclin-dependent kinases (CDK) 4 and 6 forming a complex that phosphorylates and thus activates molecules that allow cell cycle progression from G1 to S phase promoting cell proliferation. Moreover, cyclin D1 sequesters inhibitors of CDK kinases such as p27.

Usually, the mRNA resulting from the CCND1 gene does not undergo alternative splicing; however, it has been seen that in multiple cancers and tumors there are translocations and deletions that promote the activation of this gene (49). In PCa, it has been determined that a single nucleotide polymorphism for this gene is common, this allele called G/A870 consists of the change of a guanine by an adenine in the vicinity of the end of exon 4 and beginning of intron 4. This mutation causes a decrease in the recognition of the usual splice sites and favors the formation of a transcript that retains a fraction of intron 4 that includes a premature stop codon, generating the isoform called cyclin D1b ( Figure 1E ). This isoform always resides in the nucleus, unlike the normal isoform that shuttles between the nucleus and the cytoplasm according to the signals in the cell. The expression of this isoform also correlates with a decrease in the activation of the androgen receptor in PCa (23).

CD44 is an adhesion glycoprotein expressed in the epithelial cells of prostate tissue. The CD44 gene consists of 20 exons, where intermediate exons v2 to v10 are mutually exclusive and could be included due to alternative splicing decisions. It has been reported that during PCa a deregulation of this alternative splicing selection occurs, generating various isoforms at different stages of the disease. However, it has been observed that in metastatic stages of PCa there is a negative regulation in the expression of this glycoprotein while migration is decreased (24). One report proposes that CD44v6 is expressed in epithelial to mesenchymal transition (25). Considering this evidence, CD44 and CD44v6 could be an interesting marker to find prostatic metastatic cells in liquid biopsies.

The KLK3 gene encodes the protein peptidase related to kallikrein 3 (KLK3), better known as the prostate specific antigen (PSA). KLK3 functions as a protease and it plays an extremely important role during the development of PCa, where it is overexpressed in order to degrade the extracellular matrix and thus expand the cancerous tissue (26). Moreover, an increase in angiogenesis also correlates with the overexpression of KLK3 in the context of PCa (27).

KLK3 consists of 5 exons and 8 isoforms of the KLK3 protein are known. Even when different attempts have been made in order to relate the different isoforms of KLK3 with malignancy and progression of PCa, no irrefutable evidence has been found yet and it becomes interesting to explore alternative splicing regulation for this PCa biomarker (26).

Both the CD44 and KLK3 genes are important in the development of PCa disease and although they are well studied, alternative splicing events are highly complex, and not only have polymorphisms been reported that modify the dynamics of splicing, It has also been seen that this modification can be orchestrated by epigenetic mutations and therefore are not very useful as a therapeutic target. Interestingly, there was a sharp decline in the overall incidence of PCa from 2007 to 2014 in the US, which correlated with a reduction in prostate specific antigen (PSA) screening as a result of changes to US Preventive Services Task Force recommendations. Variations in PCa incidence rates mainly reflect the use of PSA screening, which typically detects localized disease. In 2023, it was estimated that 55% of American men with metastatic PCa were initially diagnosed with localized or regional disease, reflecting the importance of PSA detection. Even when the full impact of splicing decisions for CD44 and KLK3 in the onset an evolution of PCa remains elusive, it results interesting to analyze the expression of splicing variants during the progression of the disease in order to develop new and improved diagnostic and therapeutic tools.

FOXA1 is a transcriptional factor responsible for the modulation of key splicing factors such as hnRNPK and SRSF1 (50). Although several mutations have been detected in the FOXA1 gene, the effect of these mutations on the expression of genes regulated by splicing factors including SRSF1 (like AR or VEGFA) has not been fully understood yet. The FOXA1 gene has been found to have a very high mutagenesis rate, which is reflected in the ability to generate tumors in basically any endoderm-derived tissue, including the prostate gland (28).

The relationship between FOXA1 and splicing regulation in the context of PCa is still not fully understood (29), but we believe that the analysis of this cellular cascade in the following years could mean a significant advance for the development of diagnostic targets.

Insulin-like growth factor I (IGF-I), also called somatomedin C, is essential in developmental and proliferative functions in many tissues, besides maintaining homeostasis of several hormones including insulin. This factor is secreted mainly by the liver and some other tissues such as the prostate in a paracrine manner.

The gene encoding this factor (IGF-I) consists of 6 exons that generate 3 different isoforms: IGF-IEa, IGF-IEb and IGF-IEc (30) and the IGF-IEc isoform is highly expressed during PCa and this has been directly related to an increase in proliferation mediated by the activation of the IGF-1R receptor in PCa epithelial cells ( Figure 1C ). Interestingly, several studies have shown an association between increased levels of circulating IGF-1 and the risk of developing solid malignancies, including PCa (31). The IGF-IEc isoform is overexpressed in human PCa tissues and in human PC-3 and LNCaP cells. This preferential IGF-IEc mRNA expression generates the MGF E peptide that possesses mitogenic activity through mechanisms independent of IGF-1R, IR, and hybrid IGF-1R/IR (51). Altogether, these observations suggest that IGF-1 and IGF-IEc could act as circulating biomarkers for PCa diagnosis.

There are also reports of mutations that favor the formation of other IGF-I isoforms, although they have not yet been related to PCa.

The KLF6 gene (krüppel-like transcription factor 6), consisting of 3 DNA-binding zinc domains, regulates the cell cycle by inducing the expression of the cell cycle inhibitor p21 and preventing replication as its main function; however, KLF6 is a factor that can also be one of several modulators of cell differentiation, apoptosis and development and is therefore considered a tumor suppressor.

Usually, KLF6 is expressed without undergoing alternative splicing (wtKLF6 isoform). However, individuals possessing the IVSΔA allele generate 3 alternative isoforms: KLF6-V1, KLF6-V2 and KLF6-V3. These isoforms lack one (KLF6-V2) or 2 zinc domains (KLF6-V1 and KLF6-V3) and therefore are not functional ( Figure 1D ). In addition, alternative variants can also act as antagonists of the functional isoform wtKLF6 in such a way that it behaves as a dominant allele and therefore cancers related to this isoform tend to be aggressive and generate metastasis. The IVSΔA allele possesses a unique G/A polymorphism that generates a new splice site near the boundary between the first intron and the second exon (called IVS1 -27 point) that is recognized by the SRp40 protein and promotes the expression of isoforms lacking zinc domains (32).

The presence of the IVSΔA allele has been studied only in the American population and it has been determined that it is related to lung and PCa in men (33).

The ERG gene (ETS-related gene) is a member of the E-26 transformation-specific (ETS) family of transcription factors. The ETS transcription factors have a pivotal role in development and cell differentiation with roles in different cell types, where it regulates embryogenesis, vasculogenesis, angiogenesis, haematopoiesis and neuronal development. This transcription factor regulates the expression of genes involved in the regulation of cellular architecture, cell migration, invasion and cell permeability.

There are several descriptions of ERG’s gene and exon/intron structure. The ERG locus is approximately 300 kb long and includes at least 12 exons producing up to 30 alternative ERG transcripts that are expressed and encoding at least 15 protein variants ( Figure 1F ).

Prostate epithelia do not normally express ERG, but it has been reported that ERG is overexpressed in a high proportion of prostate carcinomas as a result of a gene fusion with the androgen-driven promoter of the TMPRSS2 gene. This fusion is caused by chromosomal translocation or by interstitial deletion of the intergenic region between TMPRSS2 and ERG (34). Deletions may occur because of fragile sites and breakpoints found in intron 2 of ERG and in introns 1 and 2 of TMPRSS2 that resemble Alu repeat elements. These elements are involved in gene expression at different levels, including alternative splicing. This aberrant ERG represses a number of prostate epithelium-specific genes, including KLK3, suggesting that ERG promotes the de-differentiaton of prostate epithelium (52).

Another alternative splicing decision relevant for ERG expression in prostate cancer involves cassette exons 7 and 7b. It has been reported that when exon 7b is included, ERG is phosphorylated by ERK kinase, triggering the posttranslational activity that drives cell proliferation (35).

It has been seen that this ERG/TMPRSS2 deletion is present in approximately 50% of PCas and it has been demonstrated that inhibiting the insertion of exon 7b decreases cell proliferation and migration. Due to the complexity of this gene, no trans acting splicing regulatory factors have been described yet but the development of tools designed to modify these splicing events are attractive (36).

The transcription factor NF-Y supports cell proliferation by activating the transcription of various molecules that allow transformation and proliferation. This factor comprises 3 subunits: NF-YA, NF-YB and NF-YC. The gene coding for the NF-YA subunit consists of 9 exons where the first exon corresponds to a non-coding sequence and the third exon undergoes alternative splicing that encodes 28 amino acids ( Figure 1G ), producing the NF-YA_L and NF-YA_S isoforms, respectively (37). Although the dynamics that deregulates the expression of these isoforms is not fully known, it has been seen that other epithelial cancers such as lung adenocarcinoma overexpress the long isoform NF-YA_L which has been related to cell migration, while the short isoform NF-YA_S is related to endometrial cancer tumors. In PCa, it has been observed that the increase of NF-YA_S induces cancer cell proliferation, while NF-YA_l overexpression increases cell motility. We believe that evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients (38).

The vascular endothelial growth factor or VEGFA promotes angiogenesis and the gene that encodes this factor can generate different angiogenic isoforms that are generally referred to as “VEGFAxxx” where xxx refers to the number of amino acids present in the protein. According to this, each isoform has a different half-life and activates various signaling pathways ( Figure 1H ).

The angiogenic isoforms VEGFA121, VEGFA145, VEGFA148, VEGFA165 and VEGFA206 are normally expressed in cells but they show a certain ratio with the anti-angiogenic isoforms (VEGFA121b, VEGFA145b, VEGFA148b, VEGFA165b, VEGFA206b), so the regulation of their expression is essential for the correct functioning of the tissue (39). However, in PCa cell lines it has been found that the ratio between these pro-angiogenic and anti-angiogenic isoforms is affected and expression tends to be enhanced for the pro-angiogenic isoforms (40). As in the case of the androgen receptor (AR), this change in isoform regulation is not related to a mutation in the VEGFA gene so far, but it is the dysregulation of the SRSF1 factor that mainly increases the expression of VEGFA121 and decreases the isoform with the best anti-angiogenic potential (41).