2.1 Molecular structure of DIAPH3

The full-length DIAPH3 protein features several key structural domains: an N-terminal GTPase-binding domain (GBD, also called FH3), a diaphanous inhibitory domain (DID), a dimerization domain, a coiled-coil region, and two FH domains (FH1 and FH2), followed by a C-terminal diaphanous autoinhibitory domain (DAD) (Figure 1A) [3]. DID is part of the FH3 module. In the prototypical formin structure, the N-terminal DID interacts with the C-terminal DAD, resulting in an auto-inhibitory state [9]. Upon binding to Rho GTPases (such as RhoA, RhoC, Rif, and Cdc42), the GBD alleviates this auto-inhibition, allowing for a conformational change that exposes the functional domains FH1 and FH2. The FH1 domain, located at the N-terminus of the FH2 domain, contains multiple contiguous proline residues that serve as binding sites for the actin monomer binding protein Profilin. By enhancing delivery of new actin monomers to the growing actin filaments, Profilin thereby accelerates filament elongation [10]. Meanwhile, the FH2 domain, consisting of approximately 400 amino acid residues, promotes the incorporation of actin monomers into the barbed end of the growing non-branched actin filament, ultimately enabling the bundling of actin filaments [3]. The mRNA and protein sequences of various DIAPH3 isoforms generated through alternative splicing have been reported in the NCBI and Swiss-Prot databases (Figure 1B). DIAPH3 facilitates isoform-selective actin assembly, which plays distinct and differentially regulated roles during cell adhesion and motility. Notably, isoform-a and isoform-f are the largest and most prevalent isoforms expressed in human tissues, with isoform-f being specifically associated with filopodia formation. In contrast, isoform-b is uniquely involved in the process of plasma membrane blebbing [5]. Such diversity in gene-structure associated functions highlights a complex regulatory mechanism by which DIAPH3 isoforms contribute distinctly to cellular dynamics.

2.2 DIAPH3 working models

Extracellular cues trigger a reorganization of the cytoskeleton by stimulating cell surface receptors, including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and integrins. For instance, the mitogen lysophosphatidic acid (LPA) activates specific GPCRs, leading to the activation of various guanine nucleotide exchange factors (GEFs) [11]. These GEFs, in turn, regulate the activity of RhoA GTPases. The formation of specific actin-based structures by DIAPH3 is likely dependent on the GTPases and their downstream effectors. For example, the RhoA GTPases Rif and Cdc42 activate DIAPH3 to promote the formation of filopodia. Additionally, other GEFs can activate the Rho GTPases RhoB and RhoD, which localize to the membrane of endosomes, and subsequently stimulate the activation of DIAPH3 [8].

Once the structural auto-inhibition of DIAPH3 is relieved, it fulfills its role in F-actin assembly and participates in various other cellular functions that rely on the actin cytoskeleton (Figure 2A). For instance, DIAPH3 is crucial for E-cadherin dependent adhesion, thereby maintaining the integrity of multicellular epithelial structures [12]. Additionally, DIAPH3 is indispensable for the process of cytokinesis [13]. DIAPH3 localizes to the cytokinetic furrow, where it contributes to the formation of a β-actin network. Alterations in actin isoforms can lead to cytokinetic failure [14]. Moreover, DIAPH3 can undergoes liquid-liquid phase separation (LLPS) to form condensates, resulting in spatial detainment of DIAPH3 from the F-actin assembly sites, thereby inhibiting formation of filopodia under stress [15].

Figure 2 Discovered functions of DIAPH3. (A) DIAPH3 regulates actin nucleation and polymerization. (B) DIAPH3 modulates microtubule dynamics by interacting with microtubule binding proteins. (C) DIAPH3 facilitates the release of megakaryocytic acute leukemia (MAL) into the nucleus, where it acts as a coactivator of serum response factor (SRF) to regulate the transcription of genes related to the cytoskeleton.

Interestingly, DIAPH3 is also implicated in functions that are independent of actin. A construct encompassing the FH1 and FH2 domains of DIAPH3 has been shown to induce stable microtubules without relying on its dimerization and actin nucleation properties [16]. DIAPH3 interacts with the microtubule plus-end binding protein EB1 and adenomatous polyposis coli (APC), facilitating the stabilization of microtubules at the leading edge of the cell during polarized cell migration (Figure 2B) [16]. Notably, downregulation of DIAPH3 leads to a decrease in microtubule stability, which in turn increases susceptibility to taxane drugs that inhibit microtubule dynamics [17]. Furthermore, membrane-derived microvesicles, which are regulated by microtubules, are secreted from prostate cancer (PCa) cells and have been shown to depend on DIAPH3 expression [18]. Moreover, mass spectrometry-based quantitative proteomic analysis has revealed that the interactome of mDia2 (the mouse homolog of DIAPH3) is involved in cell cycle, cell death regulation, and post translational modification, particularly in ubiquitination [19]. DIAPH3 forms a complex with FBXO3 and p53, modulating p53 activity in an actin nucleation-independent and conformation-insensitive manner. These observations raise a possibility that DIAPH3 not only acts as an actin assembly factor but also functions in various other cellular processes [19].

Emerging evidence suggests that DIAPH3 also plays crucial roles in the nucleus. It contains functional nuclear localization sequences, justifying a possible involvement in nuclear activities. Although the specific requirements for DIAPH3’s function within the nuclear environment have yet to be clearly established, recent findings highlight its potential role in transcriptional regulation. Actin polymerization alleviates the inhibition between actin monomers and megakaryocytic acute leukemia (MAL), enabling MAL to translocate into the cell nucleus. As a coactivator of serum response factor (SRF), MAL promotes the transcription of various genes associated with the cytoskeleton, including focal adhesion kinase (FAK), β1-Integrin, Talin, Zyxin, and Vinculin (Figure 2C) [20,21]. Moreover, there is growing interest in the notion that formins may influence transcription by remodeling the nucleoskeleton [22]. A recent study has shown that functional mutations in DAAM2, a member of the formin family, are associated with androgen insensitivity syndrome [23]. DAAM2 is enriched in the nucleus, where its localization correlates with that of the androgen receptor (AR), forming actin-dependent transcriptional droplets in response to androgen dihydrotestosterone (DHT). DAAM2 polymerizes actin directly at the AR to promote droplet coalescence, and nuclear actin polymerization, which is required for prostate specific antigen (PSA) expression in PCa cells [23]. Similar to DAAM2, DIAPH3 can also undergo LLPS to form comparable condensate structures [15]. Based on these findings, it is conceivable that DIAPH3 plays a role in the dynamic reorganization of nuclear structures, thereby contributing to the regulation of gene transcription.

Overall, while the precise mechanisms underlying DIAPH3’s functions in the nucleus remain to be fully elucidated, its potential involvement in these processes opens new avenues for better understanding the role of formin members in cellular dynamics.

4.1 Pan-cancer mutational landscape of DIAPH3

To systematically unravel the role and underlying mechanisms of DIAPH3 in cancer, we conducted a comprehensive analysis of the transcriptional and mutational landscape of DIAPH3 across 32 human cancer types through The Cancer Genome Atlas (TCGA) database. Gene expression analysis revealed that DIAPH3 is generally upregulated in most cancers (18 out of 24) when compared to normal counterparts, with the exception of kidney chromophobe carcinoma (KICH), where it is found to be downregulated (Figure 3A). Owing to a lack of expression data in normal tissues, certain cancer types are not included in this analysis. These data indicate an overall trend of upregulation of DIAPH3 in human cancers, suggesting, likely, an oncogenic role for it in tumorigenesis.

Figure 3 Transcriptomic and mutation landscape of the DIAPH3 gene across 32 human cancers. (A) Pairwise comparison of DIAPH3 expression between normal and tumor tissues in the indicated TCGA cancer types. (B) Bar plots illustrating the cumulative aberration frequencies of DIAPH3 associated with the indicated genetic alterations. Data derived from TCGA pan-cancer analysis encompassing 10,953 patients representing 32 cancer types were viewed through cBioPortal. Adrenocortical Cancer (ACC); Bladder Cancer (BLCA); Breast Cancer (BRCA); Cervical Cancer (CESC); Bile Duct Cancer (CHOL); Colon Cancer (COAD); Diffuse Large B Cell Lymphoma (DLBC); Esophageal Cancer (ESCA); Glioblastoma (GBM); Head and Neck Cancer (HNSC); Kidney Chromophobe Carcinoma (KICH); Kidney Clear Cell Carcinoma (KIRC); Kidney Papillary Cell Carcinoma (KIRP); Acute Myeloid Leukemia (LAML); Lower Grade Glioma (LGG); Liver Cancer (LIHC); Lung Adenocarcinoma (LUAD); Lung Squamous Cell Carcinoma (LUSC); Mesothelioma (MESO); Ovarian Cancer (OV); Pancreatic Adenocarcinoma (PAAD); Pheochromocytoma & Paraganglioma (PCPG); Prostate Adenocarcinoma (PRAD); Rectum Adenocarcinoma (READ); Sarcoma (SARC); Skin Cutaneous Melanoma (SKCM); Stomach Cancer (STAD); Testicular Cancer (TGCT); Thyroid Cancer (THCA); Thymoma (THYM); Uterine Corpus Endometrial Carcinoma(UCEC); Uterine Carcinosarcoma (UCS); Ocular melanomas (UVM).

Genomic alterations are often linked to gene dysregulation. To systematically uncover the underlying mechanisms of DIAPH3 misexpression, we surveyed the mutational landscape of DIAPH3 genes across 32 human cancers. Results identified a collectively low mutation frequency in human cancers, with an average mutation rate of only 3.2% (Figure 3B). This rate varies among different cancer types, ranging from 0.2% in thyroid carcinoma (THCA) to 8.32% in uterine corpus endometrial carcinoma (UCEC). Mutations were identified as a primary form of genomic alterations, particularly in cancers such as UCEC, skin cutaneous melanoma (SKCM), and lung adenocarcinoma (LUAD). This overall very low mutation rate indicates a limited contribution of genomic alteration to the global transcriptomic dysregulation of DIAPH3. Epigenetic modifications may thus play a crucial role in DIAPH3 misexpression. Studies have shown that the super enhancer-regulated long non-coding RNA (LncRNA) LINC01089 is involved in the downregulation of DIAPH3 expression [30]. LINC01089 binds to heterogeneous nuclear ribonucleoprotein M (hnRNPM), thereby enhancing hnRNPM-mediated skipping of DIAPH3 mRNA exon 3. This specific exon 3 contains a significant m6A-modified site that can be recognized by IGF2BP3, consequently promoting DIAPH3 mRNA stability. By skipping exon 3, the expression of DIAPH3 is subsequently reduced [30]. Further studies examining the mechanisms driving DIAPH3 dysregulation are key to dissecting the complex role of DIAPH3 in cancer biology.

4.2 DIAPH3 in control of tumor progression

In the past few decades, there has been a growing focus on the involvement of DIAPH3 in tumor progression, spanning from primary tumor development to metastasis. DIAPH3 has been reported to promote the proliferation of cervical cancer cells by activating the mTOR signaling pathway [31]. Additionally, overexpression of DIAPH3 in lung cancer enhances tumor growth by weakening the interaction between STK38 and MEKK, resulting in an activation of the mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) pathway (Figure 4A) [32]. Notably, significant attention has been directed towards its role in cancer cell migration, invasion, and metastasis.

Figure 4 DIAPH3 signalosome in tumor progression. (A) High DIAPH3 expression in lung cancer promotes cancer cell proliferation via the MEK/ERK signaling pathway. (B) Overexpression of DIAPH3 enhances its interaction with HSP90, inhibits the binding of HSP90 to GSK3β, and activates the β-catenin/TCF signaling pathway in hepatocellular carcinoma. (C) Activin A signaling induces the upregulation of DIAPH3, which further contributes to tumor development by reducing the nuclear pool of p53 and promoting the expression of carcinoma-associated fibroblasts (CAFs) marker genes, as well as fibroblast proliferation and migration. Arrows indicate promotion, while perpendicular lines denote inhibitory effects.

4.3 Controversial role of DIAPH3 in PCa

Altered expression of DIAPH3 has been frequently observed in human cancers and has been causally linked to tumorigenesis. Examples of the roles played by DIAPH3 in cancers are summarized in Table 1. While DIAPH3 is often found to be protumorigenic, promoting progression in cancers such as lung [32], breast [42] and pancreatic [45] cancer, there are also instances where it might suppress tumor progression. Particularly, there are reported conflicting roles of DIAPH3 within a specific cancer type. Next, we dissect such situation using PCa as a prime example. An earlier study in 2012 [38] reported that DIAPH3 is downregulated or even completely lost in some samples of prostate, liver, and breast cancers. Further experiments demonstrate that silencing DIAPH3 in Ras-overexpressing mammary epithelial cells (HMEC) and DU145 PCa cells significantly enhances their migratory and invasive capabilities, as well as their potential for distant metastasis. While these results imply a potential tumor suppressor role for DIAPH3, it is crucial to acknowledge the limitations of the study, which utilized a single set of expression data from a PCa cohort with a small sample size (4 normal vs. 16 tumor tissues). Additional data analysis of a different PCa cohort indicated a relationship between the loss of DIAPH3 copy number and a higher Gleason score. However, as previously discussed, the frequency of mutations and deletions involving DIAPH3 in cancers is globally very low, and a reduction in copy number does not always correspond to a decrease in its mRNA levels.

Table 1 DIAPH3 functions in different cancer types.

On the other hand, analysis of patient data from the largest TCGA database reveals a contrasting picture, where DIAPH3 is significantly overexpressed in PCa (Figure 3A). This elevated expression is positively correlated with tumor malignancy and is associated with a poor prognosis for PCa patients [38]. Additionally, some basic bioinformatic analyses of DIAPH3 have been conducted in pan-cancer studies. Specifically, gene enrichment assays in PCa have shown that DIAPH3 expression is negatively correlated with the p53 signaling pathway and tumor inflammation, while positively correlated with tumor proliferation, Myc target genes, angiogenesis, and the TGF-β signaling pathway. Tumor immune estimation resource (TIMER) analysis has also indicated that DIAPH3 expression in PCa is negatively correlated with immune responses and the infiltration of various immune cells, including B cells, natural killer (NK) cells, T effector cells, and macrophages [46]. These findings suggest that DIAPH3 plays an oncogenic role. Such discrepancy highlights a need for detailed studies to elucidate the precise role and mechanism of DIAPH3, in order to devise novel therapeutic targets against aggressive PCa.

5.1 DIAPH3 as a prognostic biomarker

Due to its critical roles in cancer, the potential of DIAPH3 as a prognostic biomarker or therapeutic target has been actively pursued. We conducted a systematic LASSO Cox regression analysis on the expression levels of DIAPH3 to construct a prognostic model. Our findings suggest that, in most cases, DIAPH3 is linked to an unfavorable prognosis (Figure 5A), which is consistent with its elevated expression in the majority of cancer types (Figure 3A). Specifically, high levels of DIAPH3 mRNA correlate with poor outcomes in several cancers, including adrenocortical carcinoma (ACC), KICH, pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), and uveal melanoma (UVM). Further Kaplan–Meier (KM) survival analysis demonstrated that an abnormal elevation of DIAPH3 expression in ACC, PAAD, and Lower-Grade Glioma (LGG) serves as a detrimental prognostic factor for cancer patients (Figure 5B). Some studies have also examined the relative expression of DIAPH3 at the mRNA level and/or at the protein level through immunohistochemical analysis of tumor versus normal tissues to identify associations with the clinicopathological characteristics of the cohorts. For example, an abnormal increase in DIAPH3 levels has been observed in cervical cancer, where it has been identified as an unfavorable prognostic factor [46]. In contrast, in glioblastoma, elevated DIAPH3 expression, coupled with an increased proliferation rate, is linked to longer survival, particularly among patients with O6-methylguanine-DNA-methyltransferase (MGMT) methylation [47]. These results underscore the potential of DIAPH3 as a valuable biomarker in cancer prognosis, warranting further investigation into its role in cancer biology.

Figure 5 The clinical relevance of DIAPH3. (A) Least absolute shrinkage and selection operator (LASSO) regression analysis of the DIAPH3 gene in 33 human cancers. The hazard ratio of DIAPH3 in each cancer is illustrated in the figure. (B) Kaplan–Meier plots showing DIAPH3 as an unfavorable gene associated with patient overall survival in the indicated cancer types. Data derived from TCGA pan-cancer analysis encompassing 10,953 patients representing 33 cancer types were viewed through cBioPortal. Gene expression and survival analysis were visualized via the online database UALCAN (http://ualcan.path.uab.edu/index.html).

5.2 DIAPH3 as a therapeutic target

Currently, there are no commercially available DIAPH3-specific inhibitors. However, a small molecular inhibitor of formin FH2 domains, known as SMIFH2, has been identified as an effective inhibitor of formin-mediated actin assembly through a screening of 10,000 commercially available small molecule compounds in vitro [50]. Biochemical analyses indicate that the evolutionarily conserved FH2 domain serves as the molecular target of SMIFH2. Its inhibitory action is specific, affecting both the nucleation and elongation of actin, which are mediated exclusively by formins. Recent studies suggest that SMIFH2 exhibits similar inhibitory effects on six of the seven classes of formins in mammals, including DIAPH3 [51], indicating its broad-spectrum potential against formin-family-mediated actin assembly. In renal epithelial cells, treatment with SMIFH2 resulted in a reduction of filamentous actin [52]. Additionally, in U2OS and HCT116 cells, SMIFH2 treatment disrupted the cycles of actin and microtubule depolymerization and repolymerization, adversely affecting the structure of the Golgi apparatus [53]. The Dia family is widely recognized as a key regulator of the cytoskeleton involved in tumor invasion, and targeting it with SMIFH2 is considered a promising therapeutic approach (Table 2). In experiments with epithelial ovarian cancer cells (SKOV3 and ES2), treatment with SMIFH2 significantly inhibited monolayer cell proliferation. Moreover, combining SMIFH2 with paclitaxel exhibited an additive effect in suppressing the viability of ovarian cancer spheroids [54]. It is worth noting that emerging studies indicate that formins may not be the only target of SMIFH2. Notably, SMIFH2 has been shown to block interferon-induced JAK-STAT signaling [55] and downregulate the p53 protein level in a non-proteasome dependent manner, reducing its transcriptional activity [53]. These findings suggest that caution should be exercised when considering SMIFH2 as an inhibitor of formins due to potential off-target effects.

Table 2 The role of DIAPH3 agonists and antagonists in cancers

In a separate study, compounds IMM-01 and IMM-02 were identified based on their ability to activate the mDia family by disrupting the DID-DAD binding [56]. Molecular docking simulations revealed that they occupy the armadillo-repeat region of the mDia DID structure, a site typically occupied by DAD under physiological conditions [56]. Due to their differing binding orientations, IMM-01 and IMM-02 exhibit varying binding affinities and thus physiological effects. In immortalized NIH-3T3 cells, IMM-01 induced the formation of filamentous lamellipodia and stabilized microtubules, consistent with formin activation, while IMM-02 had a less pronounced effect [56]. This inhibition of microtubule disassembly may contribute to the stabilization of the cytoskeleton and the arrest of mitosis, ultimately leading to the induction of apoptosis, similar to the action of taxane-based chemotherapeutic agents [57]. Notably, continuous treatment with either compound significantly inhibited the invasion of glioblastoma U87 cells, a result achieved through mDia activation [58]. In SW480 colon cancer cells, treatment with 15 μM IMM-01 successfully reversed the persistent activation of the EGFR pathway induced by Dia depletion and inhibited tumor cell proliferation and migration [59]. Additionally, both intraperitoneal and tail vein injections of IMM-01 or IMM-02 were able to inhibit the growth of subcutaneous tumors in nude mice [56]. IMM-01 also acts as an agonist for DAAM2, another member of the formin family containing the DID-DAD self-inhibition structure. It was shown to rescue the impaired formation of lamellipodia caused by DAAM2 knockdown in human podocyte cell lines when treated with 30 μM IMM-01 [60].

Both SMIFH2 and the compounds IMM-01 and IMM-02 show promise as therapeutic agents targeting formins (Table 2). SMIFH2 demonstrates broad-spectrum inhibition of these proteins, while IMM-01 and IMM-02 specifically activate the Dia subgroup of formins, highlighting their potential for targeted cancer therapies. However, these modulators lack specificity, as they can affect the activity of multiple formins. This non-specificity raises the likelihood of side effects, emphasizing a need for search of molecules that can specifically target DIAPH3. In parallel, continued exploration of DIAPH3’s implications in cancer pathogenesis is expected to open new avenues for precision medicine.

Author contributions

J.X. and D.Z. conceived the conceptualization, scope, and outline of this review. J.X., L.H., Y.M., D.W. and D.Z. collected and analyzed the relevant references. J.Z. and C.Z. performed the bioinfomatic analysis, J.X., L.H. and D.Z. prepared the schematic illustrations. J.X., L.H., L.Y., Y.F., Y.M., D.W. and D.Z. wrote and revised the manuscript. All authors participated in critical reading and the final approval of the manuscript.

Funding

This work was supported by grants from the Shenzhen Science and Technology Program (JCYJ20220530160410024 to DZ and GJHZ20220913144209018 to DW), Guangdong Basic and Applied Basic Research Foundation (2024A1515012312 to DZ), China Postdoctoral Science Foundation (2022M721100 to JX and 2024M750867 to CZ), and the Changsha Natural Science Foundation (KQ2202154 to JX and KQ2208049 to LY).

Availability of data and materials

The material supporting the conclusion of this review has been included within the article.

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Not applicable.

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Not applicable.

Competing interests

The authors declare that there is no conflict of interests.