The WASP/WAVE Protein Family in Breast Cancer and Their Role in the Metastatic Cascade

WASPs and WAVEs

Discovery and members of the family. In 1937, Alfred Wiskott first delineated the symptoms of microthrombosis, bloody diarrhoea, eczema, pyrexia, and recurrent ear infections in three brothers while sisters were not affected. He hypothesized it is a novel hereditary thrombophilia different from atopic thrombocytopenic purpura. Later, in 1954, Robert Aldrich studied a family where 16 out of 40 males over six generations showed similar symptoms and no female cases, demonstrating an X-linked pattern of inheritance. This condition was later named in honour of Wiskott and Aldrich as the great pioneers for its identification and description (12).

In 1994, Derry et al. (12) successfully isolated and identified the gene responsible for Wiskott-Aldrich Syndrome, hence naming it Wiskott-Aldrich Syndrome Protein (WASP), mainly found in the cytoplasm of hematopoietic cells (13). Two years later, a Japanese team found a 65-kDa brain protein, homologous to WASP but with a broader expression, particularly in nerve cells, and named it neural WASP (N-WASP) (14). Further research discovered a cDNA structurally homologous to WASP and N-WASP, resulting in the designation of the resultant protein as WAVE, for WASP-family verprolin-homologous protein, marking it as the third member of the WASP family (15). Further exhaustive database analyses identified two additional mRNAs encoding proteins related to WAVE, named WAVE2 and WAVE3, thus completing the five major members of the WASP family discovered hitherto.

This tremendous research has expanded the WASP family. New identifications in recent discoveries include junction-mediating and regulatory protein (JMY), Wiskott-Aldrich syndrome protein and scar homolog (WASH), and WASP homolog associated with actin, membranes, and microtubules (WHAMM). Also, among the recent discoveries are a couple of proteins, WASP and MIM-like (WAML) and WASP lacking the WH1 domain (WAWH). Due to the evolving nature and partially understood science of these new proteins, their mention will be brief in this regard (16, 17).

Structure

Members of the WASP family possess a common proline-rich domain (PRD) as well as a VCA region at the end of their C-terminus. Each individual within this family exhibits a distinct N-terminus (Figure 2) (18). WASP and N-WASP exhibit substantial structural similarity, sharing 50% homology. Both possess identical functional domains and motifs, such as the EVH1 (or WH1) domain, a highly basic region, a GBD/CRIB motif, a proline-rich region, and a VCA region (19).

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Figure 2.

The protein domains of individual members of the WASP family. The C terminus is highly conserved between the members, while the N terminus is divergent. WH1, WASP homology domain1; WHD, WAVE homology domain; WMD, WHAMM membrane interaction domain; WAHD1/2, WASH homology domain 1 and 2; LIR, LC3-interacting region; B, basic domain; GBD, GTPase binding domain; CC, coiled-coils domain; PPP, polyproline domain; V, verprolin homology domain; C, central domain; A, acidic domain. In general, the N-terminal domains are thought to confer spatiotemporal regulation on the protein, while the WCA regions at the C-terminal are required for activation of Arp2/3.

In mammalian cells, the EVH1/WH1 domain interacts with proteins from the WASP-interacting protein (WIP) family, including WIP, CR16, and WICH/WIRE (WIP- and CR16-homologous protein/WIP-related). WASP and N-WASP predominantly form stable complexes with WIP-family proteins, enhancing their stability, protection from degradation, and overall activity (20, 21). The basic region and GBD/CRIB motif of WASP/N-WASP facilitate activation by binding to PIP2 and active Cdc42, respectively. Numerous SH3 domain-containing proteins, such as Grb2/Ash, Nck, and WISH, interact with the proline-rich area, further promoting their activation (22). The C-terminal VCA region, comprising verprolin-homology (V), cofilin-homology (C), and an extremely acidic (A) area, is crucial for initiating Arp2/3-dependent actin polymerization in vitro. The V and A regions attach to actin monomers and Arp2/3, respectively. The C region interacts with Arp2/3, causing notable alterations in its tertiary and quaternary structures, thereby influencing its capacity to initiate actin polymerization (23).

Interestingly, N-WASP features two V-structural domains, in contrast to the single V-structural domain in WASP and WAVE. This distinction potentially explains N-WASP’s enhanced efficacy in actin polymerization activation (24).

The WAVE protein, differing from WASP and N-WASP, lacks a GBD/CRIB motif. Instead, it harbours a WHC/SHC motif at its N-terminus, in addition to a fundamental domain, a domain rich in proline, and a domain VCA. Its C-terminus shares significant similarities with proteins in the WASP family (25).

Activation of the WASP/WAVE Family

WASP and N-WASP exhibit autoinhibition, but WAVE is kept inactive by a combination of four additional proteins. WASP and N-WASP are folded through an interaction between the GBD/CRIB domain and the cofilin-homology (C) domain, which conceals the VCA region, and prevents the ARP2/3 complex from accessing the VCA region (8). Rac and Nck activate WAVE by disassembling the regulatory complex, so allowing WAVE to activate the Arp2/3 complex (26).

Where the C-terminal VCA domain binding to the Arp2/3 complex promotes its WASP activation and finishes the nucleation event, initiating actin polymerization and branched-actin networks assembly, the GBD domain in its N-terminal initially binds an activated Cdc42 or Rac1 GTPases converting it to an open conformation which presents the VCA domain This enables the V-domain to bind with the G-actin monomer, and the A-domain binds with the actin filament, and C-domain bind with the Arp2/3 complex within the VCA domain. Eventually, this promotes new actin filament nucleation through the conformational change carried out by the ARp2/3 complex (8, 27, 28).

Other WASP regions regulate the interaction of the VCA domain with the Arp2/3 complex. The BR domain interacts with negatively charged phospholipids at its N-terminus. The interaction includes phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 also activates WASP. The PPP domain binds to various signalling proteins, including SH3 domain-containing proteins, which recruit WASP to signalling complexes and regulate its activity (28). The activation of N-WASP is almost the same as that of WASP due to structural similarity (Figure 3) (29, 30).

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Figure 3.

WASP and N-WASP activation and their regulatory mechanism. (A) At resting state, WASP/N-WASP exist in an autoinhibited closed conformational state due to the interactions between members of the N-terminally located WASP homology domain (WH1) and the C-terminus. (B) the binding of Cdc42 to the GBD domain and the phosphoinositides (PIP2) to the WH1 domain exposes the VCA domain, the simultaneous binding of actin monomer (G-actin), and Arp2/3 complex activates Arp2/3 complex. The binding of the SH3 domain to the proline-rich region is important for Arp2/3 complex activation; (C) the active Arp2/3 complex leads to the activation of WASP/N-WASP; (D) the active WASP/N-WASP promotes promote the formation and elongation of actin filaments, contributing to actin polymerization.

WAVE proteins are regulated within a pentameric complex known as the WAVE regulatory complex (WRC) (31), it consists of WAVE itself, the specifically Rac1-associated protein 1/cytoplasmic FMR1 interacting protein 1 (Sra1/CYFIP1) subunit, Nck- associated protein 1 (Nap1) subunit, Abl-interacting factor (Abi) subunit, hematopoietic stem/progenitor cell protein 300 (HSPC300) subunit, and Nck-associated protein 1 subunit. In contrast to the activation mechanisms observed in WASP and N-WASP proteins, WAVE operates through a unique pathway significantly influenced by its interaction with Rac1, a small GTPase. The binding of Rac1 to WAVE triggers a series of conformational alterations within the WAVE regulatory complex (WRC), crucially leading to the unveiling of the verprolin-cofilin-acidic (VCA) domain. This exposure is critical for the activation of the Arp2/3 complex, thereby catalysing the initiation of actin polymerization (32, 33).

Central to this activation paradigm is the role of IRSp53, known as insulin receptor substrate p53, which fulfils a bifunctional purpose. Initially, it is instrumental in anchoring the WRC to the plasma membrane, a vital precondition for effective actin remodelling. Subsequently, IRSp53 serves as a molecular scaffold, enhancing the interaction between WAVE and various signalling entities, thereby fine-tuning WAVE’s function (Figure 4). Among these regulatory interactions, the phosphorylation of WAVE by members of the p21-activated kinase (PAK) family stands out as a critical modulatory step (34). Such phosphorylation not only completes the activation of WAVE but also promotes the availability of the VCA domain. These intricate layers of interaction and modification underline the sophisticated regulation of WAVE, setting it apart from the activation routes of WASP and N-WASP. The nuanced control over WAVE’s functionality is essential for the formation of lamellipodia and other actin-rich cellular structures, which are indispensable for cell motility and various cellular operations (35).

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Figure 4.

WAVE activation and its regulatory mechanism. (A) At resting state, WAVE exist in an autoinhibited closed conformational state. (B) the N-termini containing the WHD domain that forms a protein complex by binding to Abi, HSPC300, Nap1and Sra1.The C-termini-containing VCA domain. IRSp53 recruits the WAVE complex to the plasma membrane. Upon Rac binding to the WAVE complex, the VCA domain of WAVE is exposed, making it binding and activating the Arp2/3 complex; (C) the active Arp2/3 complex activates WAVE; (D) the activated WAVE contributes to actin polymerization and leads to the formation of lamellipodia.

WASP/WAVE Protein Family in Cell Motility

The mobility of eukaryotic cells, a cornerstone for an array of critical physiological functions, is closely tied to the dynamic reconfiguration of the actin cytoskeleton (36). This framework of filamentous proteins is essential for sustaining cell shape, providing mechanical reinforcement, and facilitating motility (37). Its leading role is evident in essential biological processes such as wound healing, immune system responses, and, most notably, in the progression of cancer metastasis, where the actin cytoskeleton’s flexible nature enables cells to rapidly adapt to external cues and navigate complex environments effectively (38). Central to the organization of this cytoskeletal flexibility are the WASP and WAVE protein families. These entities play a key role in the generation of branched actin networks, interacting with the actin-related protein 2/3 (Arp2/3) complex, an interaction crucial for the development of lamellipodia and other cellular structures imperative for cell migration and the invasion of surrounding tissues (Figure 5) (26). Through facilitating these interactions, the WASP and WAVE families not only underpin cellular structure but also drive the sophisticated cellular behaviours necessary for navigating and adapting within the cellular landscape, showing their indispensable roles in modulating actin dynamics vital for a myriad of cellular processes and pathological conditions. In 2016, Weeks et al. (39) provided further insights into the functional roles of WAVE1 and WAVE3 in actin polymerisation and cancer metastasis. Using metastatic prostate cancer cells, they demonstrated that both WAVE isoforms interact with the ARP2/3 complex to regulate actin dynamics, contributing to the metastatic phenotype. Notably, WAVE3 knockdown significantly suppressed cell growth and enhanced tyrosine phosphorylation of ARP2, emphasising its regulatory role in ARP2/3 complex activity. Interestingly, suppression of cell invasion caused by WAVE1 knockdown was rescued by ARP2/3 inhibition, highlighting functional distinctions between the two WAVE isoforms.

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Figure 5.

Molecular factors involved in cell migration. Schematics illustrate the WASP protein family in actin filament organization at the leading edge of a cell, which is critical for cell migration. WASP and N-WASP, activated by the Rho GTPase Cdc42, facilitate the Arp2/3 complex’s actin nucleation activity. Results in branched actin networks that propel cellular structures like filopodia and lamellipodia forward, driving cell movement. This process is vital for various cellular functions, including cell migration and wound healing.

In environments such as the ECM or on culture dishes, where cells adhere and navigate in a bi-dimensional manner, the intricate mechanics of locomotion are predominantly reliant on the formation of lamellipodia and filopodia at the cell’s leading edge (40, 41). Lamellipodia, characterized by their broad, leaf-like projections, are densely packed with branched actin filaments, a structure decisive for environmental sensing and propelling the cell forward (42). Conversely, filopodia are narrow, finger-like extensions enriched with tightly bundled actin filaments, acting as essential sensory tools. They excel in sensing and responding to chemical and physical cues from the ECM, thereby directing cellular migration and enhancing adhesion (43). The emergence of lamellipodia at the vanguard of migrating cells is fundamental to two-dimensional cell movement. Within these protrusions, actin branching, mediated by the Arp2/3 complex, forms a scaffold that advances the plasma membrane, propelling the cell (44, 45).

Although filopodia may not serve as the primary propulsion mechanism in cell motility, their role in mediating cell-environment interactions is critically important. Acting as cellular sensors, filopodia gauges external cues, and guides directional movement and adherence to the ECM (46). The rigid bundles of actin within these structures enable precise environmental sensing, highlighting their significance in complex processes such as tissue regeneration and the infiltration of cancer cells (47). The structural variation between lamellipodia and filopodia originates from their unique organization of actin filaments. Lamellipodia feature a dense network of actin filaments, initiated by the Arp2/3 complex, that supports the formation of a broad, advancing membrane front. This lattice is subject to continuous alteration, mirroring the cell’s locomotive adjustments (48). In contrast, filopodia are formed by tight, aligned actin bundles, spurred by linear polymerization driven by formins and other actin-interacting proteins, providing the structural rigidity necessary for environmental exploration (49). The synergistic functions of lamellipodia and filopodia are essential for efficient navigation in planar spaces. Lamellipodia provide the necessary forward momentum, while filopodia guide the direction through environmental sensing (50).

The diverse functionalities of the WASP/WAVE protein family are intricately determined by the cellular environment and external stimuli, showcasing their involvement in a spectrum of cellular activities ranging from phagocytosis in immune cells to maintaining the morphology and structural integrity of epithelial cells (51, 52).

In three-dimensional matrices that emulate the complexity of in vivo environments, the significance of WASP/WAVE family proteins is magnified, particularly in cancer cell migration. These proteins are fundamental in arranging the formation of cellular extensions capable of navigating the ECM (53, 54). WASP/WAVE proteins also can construct actin-dense structures such as invadopodia, which are imperative for the degradation of and passage through the dense ECM. Through the regulation of actin polymerization and organization, these proteins not only facilitate the physical movement of cancer cells but also enable the cells to alter their immediate microenvironment, thereby promoting metastatic dissemination (Table I). WAVE2, in particular, is vital for lamellipodia formation across different cell types, with its deficiency significantly impairing these structures’ development and, by extension, cell motility (8, 11).

Furthermore, the contribution of the WASP/WAVE family goes beyond merely aiding cell migration and invasion. These proteins are crucial for dynamic changes in cell shape and adherence, regulating the actin cytoskeleton. Through this regulation, WASP/WAVE proteins not only control the mechanical aspects of cellular movement but also impact signalling pathways involved in gene expression, cellular survival, and proliferation (55, 56).

WASP/WAVE Protein Family in Cancer Cell Protrusions

Podosomes and invadopodia are specialized cellular structures decisive for the invasion and movement of cells through the extracellular matrix (ECM). These structures are key in breaking down the ECM, enabling invasive cells to navigate through tissue barriers. Found in a variety of cell types, including monocytic lineages, smooth muscle cells, and endothelial cells, podosomes act as multifunctional mediators for cellular navigation and engagement with the ECM. Characterized by their rich actin composition, these structures exhibit rapid cycles of assembly and disassembly, underscoring their role in cellular adhesion, signal transduction, and the exertion of mechanical forces (57). Such dynamic attributes are indispensable for the processes of cell migration and ECM interaction, as delineated in the research conducted by McNiven and Baldassarre (58). This suggests the critical function of podosomes and invadopodia in cellular dynamics and tissue invasion, stressing their importance in cellular motility and interaction with the surrounding matrix.

Invadopodia, structures uniquely characteristic of invasive cancer cells, bear both structural and functional resemblances to podosomes, yet are distinctly tailored to meet the aggressive demands of cancerous behaviour. These entities extend beyond mere cellular protrusions, serving as intricate biochemical platforms where various signalling pathways converge to govern the dynamics of actin. Research by V. Artym (59) and T. Meirson (60) has emphasised the proactive role of invadopodia in the degradation of ECM components, a central action in the progression of cancer metastasis. Invadopodia enable cancer cells to traverse the ECM, promoting invasive activities and the breach of tissue boundaries.

At the heart of invadopodia lies an actin filament core, essential for both its structural integrity and function. This core is encased by a multitude of adhesion proteins and proteases, indispensable for the dismantling of the ECM and thus aiding the invasive march of cancer cells (61). Moreover, the aggregation of diverse signalling molecules within invadopodia pointed to their significance not merely as physical extensions but as vital signalling centres (62). These molecules coordinate the response to both external environmental cues and internal cellular signals, tailoring invadopodia behaviour to navigate the complex and changing landscape of the cellular environment (63).

The genesis of invadopodia, important structures in invasive cancer cells, involves a sophisticated regulatory mechanism spearheaded by N-WASP and the actin-related protein 2/3 (Arp2/3) complex. N-WASP serves as a crucial mediator in this process, orchestrating the activity of the Arp2/3 complex, which is indispensable for the initiation and elongation of actin filaments. This interaction lays the groundwork for the development of the branched actin framework that is characteristic of invadopodia (64). In seminal research by Yamaguchi et al. (65) employing siRNA techniques, it was revealed that the attenuation of N-WASP expression significantly reduces the formation of invadopodia and adversely affects the capacity for ECM degradation, indicating the vital role of N-WASP in the mechanisms of cancerous invasion. Furthering this understanding, Rotty and associates (52) provided compelling evidence of the synergistic function of N-WASP and the Arp2/3 complex in actin polymerization. Their research demonstrated that the interaction between N-WASP and the Arp2/3 complex is essential for generating the branched actin architecture, crucial for the structural integrity and functionality of invadopodia. This body of work collectively emphasizes the integral role of N-WASP in the dynamic assembly and operational efficacy of invadopodia, underlining its significance in oncogenic progression and metastasis.

WASP/WAVE Protein Family in Cancer Metastasis

The mechanisms of invasion manipulated by invadopodia and the N-WASP protein contrast with the roles of other family members like WAVE1 and WAVE2, which are deeply involved in controlling lamellipodia formation, as elucidated by Takenawa and Miki (66). This distinction within the WASP/WAVE family exemplifies the complex regulatory schemes that dictate actin cytoskeleton dynamics. Each protein’s unique function mirrors a highly sophisticated regulatory network, ensuring specific cellular processes, ranging from migration to more aggressive invasion tactics, are finely tuned by the targeted activation and interaction of these proteins. Notably, the WAVE complex, comprising WAVE1 and WAVE2, collaborates with the Rac GTPase, playing a pivotal role in lamellipodia development (67). In a divergent manner, N-WASP leverages its interaction with the Cdc42 GTPase and the ensuing stimulation of the Arp2/3 complex to lead the charge in invadopodia formation. This distinction exhibits the varied roles these proteins play in cancer cell metastasis, demonstrating their specialized functions in modulating cell movement and invasion strategies (68).

Further investigations in this field, including a significant study by Oser (69), have contributed to our understanding of the role of N-WASP in cancer dynamics. By modulating N-WASP expression across various breast cancer cell lines, this research illuminated how a reduction in N-WASP activity significantly curtails both the formation and proteolytic capabilities of invadopodia.

Recent scholarly work further delineates the paramount importance of N-WASP and the Arp2/3 complex in the trajectory of cancer development. For instance, Lomakina (70) draws attention to the adverse prognostic implications in breast cancer patients associated with diminished levels of Arpin, a member of the inhibitory Arp2/3 complex family. Additionally, Kazazian (71) unveiled a novel function of the polo-like kinase 4 (Plk4) in modulating the Arp2/3 complex, thereby influencing cell motility in cancer scenarios. This discovery suggests Plk4’s interaction with the Arp2/3 complex as a potential mechanism for altering actin cytoskeletal arrangements. In a similar vein, Laporte and Magistrato (72) employed all-atom simulations alongside dynamical network analysis to dissect the mechanisms governing Arp2/3 complex regulation. Their work lays a theoretical foundation for the development of small-molecule inhibitors aimed at curtailing cancer cell migration, presenting a novel strategy for intervention in cancer’s invasive capacities.

Targeting the N-WASP pathway has implications beyond inhibiting cancer cell movement, it can disrupt dynamic interaction between cancer cells and ECM via inhibiting the formation of invadopodia, which could affect various stages of cancer progression, from tumour invasion to metastasis (73). Understanding and combatting cancer involves integrating research findings about podosomes, invadopodia, and the N-WASP pathway for a comprehensive view of cancer cell biology. Therefore, the N-WASP pathway is not just a molecular entity of academic interest but also a promising target for developing novel cancer therapies.

WASP in Breast Cancer

Up until now, detailed insights that are specific to WASP in breast cancer are somewhat limited. The focus tends to be broader, encompassing the entire WASP family proteins, which includes N-WASP and the WAVE proteins, due to their collective involvement in actin cytoskeleton dynamics and cancer cell motility. However, the studies of Zicha et al. (74) and Ishihara et al. (75) collectively provide insight into the central roles of WASP in tumour metastasis, particularly emphasising the interplay between the immune system and cancer cells in breast cancer progression.

The tumour microenvironment (TME) constitutes a multifaceted ecosystem where cancerous cells, immune constituents, and stromal elements engage in dynamic interactions, profoundly impacting tumour evolution and the process of metastasis. Within this complex interplay, macrophages stand out due to their dualistic role that spans tumour suppression to the facilitation of tumour expansion and metastatic dissemination. Research by Zicha et al. (74) has displayed the critical function of WASP in driving macrophage chemotaxis (Table II). The study demonstrates that chemotaxis of macrophages is abolished in patients with Wiskott-Aldrich Syndrome (WAS), in these patients, while the speed of random motility of macrophages remains normal, their ability to move directionally in response to chemotactic signals is significantly impaired. This impairment is due to the disruption of the Cdc42-WASP-actin cytoskeleton pathway, The study’s findings suggest that the lack of functional WASP leads to abnormal macrophage behaviour, which may contribute to the immune deficiency and eczema observed in WAS patients.

Ishihara et al. (75) further expanded comprehension of the intricate role that WASP plays in the immune system’s contribution to breast cancer metastasis. They embarked on a study using mice that were selectively deficient in WASP within their hematopoietic cells, aiming to dissect the protein’s impact on TAMs and their capacity to aid carcinoma cell invasion and spread. The absence of WASP in these specialized mice led to a marked reduction in the ability of TAMs to facilitate carcinoma cell invasion and metastasis, demonstrating the necessity of WASP for these processes.

To explore deeper into the underlying mechanisms, the researchers engaged in ex vivo studies that involved coculturing tumour cells with macrophages lacking WASP. This approach allowed them to directly observe the motility and invasion deficits in tumour cells that result from the absence of WASP in their macrophage counterparts. Their findings enlighten the key role of WASP in driving macrophage migration towards carcinoma cells that produce colony-stimulating factor 1 (CSF-1) and in the macrophage-mediated release of epidermal growth factor (EGF) through the action of metalloproteases. This release mechanism is integral for the promotion of breast tumour metastasis, emphasizing WASP’s significant function in the intricate interplay between macrophages and carcinoma cells within the tumour microenvironment. This body of work not only elucidates a key pathway through which the immune system influences cancer progression but also identifies WASP as a critical facilitator of the metastatic process in breast cancer.

Beyond its role in immune cell function, WASP’s involvement in actin cytoskeleton remodelling suggests direct implications for breast cancer cell motility and invasion. While Ishihara focuses on the leukocyte-dependent mechanisms of metastasis, the underlying principles of WASP-mediated actin dynamics are broadly applicable to cancer cell biology. It is plausible that WASP, by regulating actin polymerization and cellular protrusions, could directly influence breast cancer cell invasion and metastatic potential.

The dual role of WASP in both the immune response within the TME and direct cancer cell behaviour presents unique therapeutic opportunities. Targeting WASP could disrupt the pro-tumorigenic activities of TAMs, potentially reverting the TME to a more tumour-suppressive state. Furthermore, inhibiting WASP function in breast cancer cells could impair their motility and invasive capabilities, curtailing metastasis. However, given WASP’s critical functions in normal immune responses and haematopoiesis, therapeutic interventions would need to be precisely targeted to minimize unintended immunosuppressive effects.

N-WASP in Breast Cancer

Martin et al. explored the impact of N-WASP in breast cancer (76). They suggested that N-WASP could act as a tumour progression inhibitor in human breast cancer (Table II). It was reported with lower relative expression of N-WASP in breast tumours versus the normal mammary tissues, and this has been associated with a reduced expression that is related to poor prognosis for the patients. Further critical support for the involvement of N-WASP in breast cancer was offered by in vitro experiments where it was demonstrated that invasive breast cancer cells with relatively higher expression of N-WASP showed reduced invasive and motile phenotype, implying its prospective clinical importance in breast cancer. Further research in breast cancer on the complex dynamics of Claudin-5, a tight junction (TJ) structure protein especially its interaction with the N-WASP signalling cascade was then done by the same team (77). In particular, they have portrayed a detailed relationship of Claudin-5 expression with balance in cell motility and adhesion using the MDA-MB-231 breast cancer cell line. Especially, the role of N-WASP during these regulatory processes was studied. Effects that were of particular interest in N-WASP inhibition in this study. Treatment of cells with a knockdown of Claudin-5 in MDA-MB-231 cells revealed that they blocked the response to N-WASP inhibition. This finding implies a direct relationship between Claudin-5 and N-WASP in signalling the motility of cells and hints at the potential role Claudin-5 may exhibit in human breast cancer metastasis through the N-WASP signalling pathway. This study evident that the N-WASP is a major regulator for the reorganization of the actin cytoskeleton and may turn out to be an attractive target for inhibition of cancer cell movement and subsequently inhibits cancer spread.

The role of N-WASP in promoting cancer invasion was further studied by Meirovitz et al., who demonstrated that triiodothyronine (T3) activates the cortactin/N-WASP/ARP2/3 signalling pathway, facilitating actin cytoskeleton remodelling and focal adhesion formation in breast cancer cells. These findings illustrate N-WASP’s critical role in translating extracellular signals into cytoskeletal rearrangements (78).

Another research utilized MTLn3 rat mammary adenocarcinoma cell lines, manipulating N-WASP expression through over-expression of a dominant-negative construct and silencing via shRNA (79). These modifications resulted in a marked decrease in the cells’ ability to form invadopodia and degrade ECM, demonstrating that decreased N-WASP activity impairs the cells’ ability to migrate, invade, intravasate, and disseminate to the lungs. The study provides substantial evidence that N-WASP is crucial for the metastatic cascade in breast cancer, particularly in the early steps of metastasis, through its role in invadopodium formation and ECM remodelling.

Although these studies indicate differing perspectives on N-WASP’s influence in regulating breast cancer cell movement, they concur on its potential association with breast cancer cell behaviour and the advancement of tumour development. The molecular mechanisms through which N-WASP operates, while still under investigation, have begun to reveal a complex network of interactions and activations. Of special relevance in this regard have been studies by Pichot et al. (68) proved N-WASP functions spanning from the activation of focal adhesion kinase (FAK) to the formation of invadopodia indispensable for the invasion of cancer cells through the extracellular matrix. N-WASP’s activation and function are modulated by oestrogen receptor-α signalling and interaction with CIP4, an F-BAR protein that facilitates its localization to membrane sites crucial for cell movement.

The period following 2015 has witnessed critical developments in elucidating the function of N-WASP in the context of breast cancer. Extensive research has augmented our insight into the molecular basis of breast cancer progression, underscoring the potential of N-WASP as a therapeutic target.

A key focus has been the role of N-WASP in promoting cancer cell invasion and metastasis. Hebbrecht et al. (80) focused on the C-terminal VCA domain of N-WASP, demonstrating how VCA nanobodies can inhibit invadopodium formation by disrupting N-WASP’s interaction with the Arp2/3 complex, thereby reducing matrix degradation activities of cancer cells. The recent study by Chung et al. (81) pointed up the emerging antiproliferative role of N-WASP in cancer biology. For squamous cell carcinoma (SCC), N-WASP particularly shows a significantly inhibitive effect concerning to proliferation and metastasis of carcinoma cells. This attenuation is mediated via an ERK2-dependent pathway, where N-WASP augments ERK2’s phosphorylation of FOXO1, subsequently leading to an increase in TXNIP expression. These interactions suggest a complex regulatory mechanism where N-WASP functions as a potential tumour suppressor.

Another critical area of research has been the relationship between N-WASP and cancer cell resistance. Recently, the role of N-WASP in breast cancer resistance to natural killer (NK) cell-mediated cytotoxicity has been increasingly recognized. N-WASP facilitates the formation of an ‘actin response’ at the immunologic synapse between breast cancer cells and NK cells. This actin response is characterized by rapid F-actin accumulation, leading to a significant reduction in the cytotoxic protease granzyme B within the target cells and thereby decreasing apoptosis rates. Knockdown of N-WASP disrupts this actin remodelling, substantially enhancing the susceptibility of previously resistant cancer cells to NK-cell-mediated lysis. The study by Al Absi et al. (82) provides compelling evidence of this mechanism, demonstrating N-WASP’s critical role in the modulation of immune evasion pathways in breast cancer.

The hormonal regulation of N-WASP has emerged as a significant area of interest. Mondaca et al. (83) highlight and discuss the molecular actions bestowed by those hormones – mainly luteinizing hormone (LH) and oestrogen – which contribute to breast cancer cell motility and invasion. These hormones in their turn generate complex pathways of signalling via their receptors bound together by the rearrangement process carried out with the actin cytoskeleton at different moments. Cortactin and N-WASP tend to be major players in both pathways during these processes. LH, as demonstrated in a study on T-47D human breast carcinoma cells, initiates a signalling cascade involving kinases like Src and FAK, leading to the activation of cortactin and the Arp2/3 complex, thus facilitating cell migration and invasion. Similarly, oestrogen’s effect, as shown in MCF-7 human breast cancer cells, entails the phosphorylation of paxillin, a protein essential for focal adhesion, which in turn regulates the N-WASP-Arp2/3 axis, modulating cytoskeletal dynamics and consequently influencing cell adhesion, migration, and invasion (84).

Decades of continuous research on Neural Wiskott-Aldrich Syndrome Protein have illustrated its potential as a therapeutic target in breast cancer, which has been increasingly recognized. N-WASP inhibitors, such as Wiskostatin, have been shown to affect various aspects of cancer progression including cell motility, invasion, and metastasis (85).

WAVE2 in Breast Cancer

Fernando et al. (86) initiated this exploration by demonstrating the elevated expression of WAVE2 in breast cancer tissues compared to normal breast tissues. Their study, using quantitative PCR and immunohistochemistry, linked high WAVE2 expression to poorer outcomes in breast cancer, specifically in node-positive cases and more advanced tumour grades. This landmark research proposed the possibility of WAVE2 as a prognostic marker for breast cancer and set grounds to launch future inquiries for its role in cancer biology. Continuing similarly, Koike and colleagues (87) clarify the predictive significance of cellular dissociation in cancer recurrence among individuals diagnosed with breast cancer. Their findings indicate that cellular dissociation, especially when associated with co-expression of Arp2 and WAVE2, is an independent significant predictor of recurrent disease. This study also emphasizes the importance of cellular morphology and molecular markers in understanding breast cancer behaviour and potential recurrence.

Expanding on the intricate dynamics of breast cancer cell behaviour, a study into the effects of pterostilbene on associated cellular mechanisms revealed the compound’s potent inhibitory action on the Rac1/WAVE2/Arp2/3 signalling axis (88). This pathway’s blockade holds considerable significance, given that the activation of the Rac1/WAVE2/Arp2/3 cascade is pivotal for the genesis of new actin filaments. The role of γ-tocotrienol on the Rac1/WAVE2 signalling pathway in mammary cancer cells detailed in another study, further expands on the theme of natural compounds in cancer treatment (89). The impact of rhapontigenin on breast cancer cells is similarly notable (90). Rhapontigenin effectively suppresses cell migration and invasion by inhibiting the PI3K-dependent Rac1 signalling pathway. This study contributes to the increasing comprehension of the impact that natural compounds can have on key signalling pathways within cancerous cells. In a study exploring the WAVE2/miR-29/Integrin-β1 oncogenic signalling axis, a new dimension of WAVE2’s role in breast cancer, particularly TNBC, is unveiled (91). Additionally, research on the complex formation between WAVE2 and PKA in breast cancer cells provides a deeper understanding of WAVE2’s involvement in cancer metastasis. This study shows that WAVE2 can act as an A-kinase anchoring protein (AKAP), recruiting PKA to specific subcellular locations, which influences the formation and enlargement of membrane protrusions essential for cell migration and invasion (92).

Very recently, Rana et al. (93) outlined the multifaceted role of WAVE2 in cancer biology by detailed review, which includes its profound effect on TNBC – well characterized for invasiveness and non-responsiveness toward currently available molecular therapies. Delving deeper into the molecular pathways regulating WAVE2, such as protein kinase B (AKT), TGF-β, VEGF, and ERK-MAPK pathways, which emphasized that the targeting of WAVE2 may be of high therapeutic potential in dealing with aggressive types of cancers.

WAVE3 in Breast Cancer

Sossey-Alaoui has been investigating the role of WAVE3 in TNBC progression and metastasis for the last 20 years, and a series of insights in this area have been published. His 2005 study brings substantial progress in understanding the role of WAVE3 in cancer metastasis (94). He investigates the complexities of WAVE3 function in cell motility and invasion, in particular of MDA-MB-231 breast cancer cells. The study employed RNA interference to knock down WAVE3 expression and observed impaired expression levels of MMP-1, MMP-3, and MMP-9, indicating a crucial role of WAVE3 in regulating these MMPs, which are key promotors in cancer cell invasion and metastasis. As a consequence, cell motility and invasion were inhibited, coupled with increased actin stress fibre formation and reorganization of focal adhesion complexes. Crucially, the suppression of WAVE3 decreased phosphor-p38 MAPK levels without affecting the levels of phospho-AKT, phospho-ERK, or phosphor-JNK. This suggests that WAVE3 influences cell motility and invasion through a pathway involving p38 MAPK but not the other MAP kinases or AKT. Following this pioneering research, his 2007 study provides an in-depth investigation into the role of WAVE3 in breast cancer progression and metastasis and reports that down-regulation of WAVE3 would significantly affect tumour growth, invasion, and metastasis (95). There has been a considerable decrease in the invasive capacities of MDA-MB-231 cells following the suppression of WAVE3. Furthermore, a noteworthy decline in the growth and spread of tumours in animal models has also been demonstrated. The study also correlates high WAVE3 expression to be related to advanced breast cancer stages, thereby indicating it might be useful in the identification of tumour progression. The same year, he demonstrated that STI-571 (Imatinib Mesylate) effectively blocks the phosphorylation of WAVE3, underscoring the essential roles of both the WAVE3-Abl kinase interaction and Abl’s kinase function in this process. This revelation identifies a precise molecular route in which Abl kinase activation of WAVE3 would enhance actin cytoskeleton dynamics, subsequently promoting cancer cell motility and invasiveness. Given STI-571’s targeted inhibition of Abl kinase, employing specific inhibitors that disrupt the WAVE3-Abl connection or directly curb Abl’s enzymatic action presents a promising strategy to thwart cancer progression (96).

Intrigued by those previous findings, in 2009, he employed quantitative RT-PCR to measure miRNA and mRNA levels, alongside Western blot analysis for protein expression and utilized luciferase reporter assays to verify the direct targeting of WAVE3 by miR200. Additionally, the impact of miR200 on cancer cell invasiveness was rigorously evaluated through cell invasion assays. The findings reveal a notable inverse relationship between miR200 expression and WAVE3 levels across various cancer cell lines, provides a comprehensive analysis of the regulatory dynamics between miR200 microRNAs and WAVE3, showcasing that miR200 directly targets and diminishes WAVE3 expression, thereby influencing cancer cell invasion and altering cell morphology (97).

Continuing on this exploration of how microRNA would influence the expression of WAVE3, the 2011 study provides a comprehensive analysis of the regulatory dynamics between microRNA miR-31 and the WAVE3 protein, in cancer invasion and metastasis (98). This work establishes a clear inverse correlation between miR-31 and WAVE3 expressions in both invasive and non-invasive breast cancer cell lines. MiR-31 directly targets WAVE3’s 3′-UTR, suppressing its expression and thereby significantly diminishing the invasive phenotype of cancer cells. This interaction not only, delineates the regulatory mechanism of WAVE3 by miR-31, positioning WAVE3 downstream of miR-31, but also emphasizes the therapeutic potential of targeting the miR-31-WAVE3 axis to mitigate cancer cell invasion and metastasis.

The comprehensive review culminating in 2012, offers a comprehensive review of WAVE3’s role in cancer progression and metastasis. It synthesizes a decade’s worth of research on WAVE3, exploring its regulation in both normal physiological and pathological conditions (99). The review details WAVE3’s involvement in actin cytoskeleton remodelling, interactions with microRNAs, and impact on cancer cell invasion. The potential application of WAVE3 as both a therapeutic target and a biomarker in breast cancer is also examined in the discussion, emphasizing its significance in subtypes of breast cancer that are characterized by aggressiveness.

Several other researchers from across the globe too have thrown light on the basic role of WAVE3 in breast cancer. Kulkarni et al. (100) have established a connection between higher levels of expression of WAVE3 and the progression and spread of TNBC. WAVE3 has been denoted as a predictive marker for cancer-specific mortality and metastatic potential, more prominently in TNBC. Taylor et al. (101) further investigate the correlation between TGF-β and WAVE3 in TNBC. They identify that TGF-β up-regulates WAVE3, associating it with the metastatic potential of TNBC cells and underscoring its role in EMT and metastasis. Both of the studies convincingly proved that WAVE3 not only contributed to the aggressive behaviour of TNBC cells but also the elevation of WAVE3 levels correlated with poor prognosis and treatment outcome. Therefore, it seems to be of utility as a therapeutic target in TNBC. Spence and colleagues (102) proceeded to advance this investigation, in which they further explored the involvement of Scar/WAVE3 in the mobility of breast cancer cells. Their research revealed that while Scar/WAVE3 is crucial for dynamic lamellipodial activities and cell motility on two-dimensional surfaces – manifested by the formation of larger, more stable lamellipodia and a reduction in cell speed – it does not significantly influence the invasive capabilities of cancer cells in three-dimensional matrices, marks the differential roles of Scar/WAVE3 in cell movement across varied environments.

Particularly, Wang et al. (103) specifically investigated the significance of WAVE3 phosphorylation in facilitating the coordination among the PI3K, TGF-β, and EGF signalling pathways. His study has indicated that WAVE3 phosphorylation is required for its oncogenic activity. It is established that WAVE3 can be phosphorylated downstream of PI3K, TGF-β, and EGF signalling pathways. This phosphorylation is not only the downstream event of these pathways but also importantly helps in its activation leading to the positive feedback loop further augmenting the progress of cancer. In vitro studies of the research showed that loss of WAVE3 phosphorylation greatly reduces migration of cells, growth of the tumorsphere, and invasion, thus showing the requirement of this post-translational modification for the behaviour of cancer cells. In vivo experiments using mouse models of breast cancer further substantiate the role of WAVE3 phosphorylation. Studies have reported that neoplasms originating from breast cancer cell lines exhibiting phosphorylatable WAVE3 (W3-WT) displayed heightened levels of phospho-AKT and phospho-SMAD in comparison to their non-phosphorylatable counterpart (W3-Y4). Consequently, this infers that the phosphorylation of WAVE3 constitutes a critical occurrence necessary for the initiation of downstream effector signalling. Only very recently, Kansakar et al. (104) probed WAVE3 phosphorylation within the proline-rich domain (PRD). They punctuated it as very important for malignancy-linked activities of WAVE3 in breast cancer, such as migration, invasion, and tumour growth and metastasis.

In 2020, a review summarizes recent advances in understanding the molecular mechanisms by which WAVE3 contributes to cancer progression (105). It discusses the regulation of WAVE3 expression and function, and the signalling pathways involved. The transforming growth factor-beta is one of the many multifunctional cytokines that determine cellular functions. In the context of cancer, this TGF-β signalling takes up a double role, that of a tumour suppressor role in the early stages, whereas at later stages of tumorigenesis promoting metastasis. Note, it has a vital role in up-regulate the expression of WAVE3 by activation of the p38 MAPK pathway which is an important signalling pathway as far as cell differentiation, apoptosis, and autophagy are concerned. This activation leads to the increased transcription of WAVE3, elevating the metastatic capability of cancer cells. The role of WAVE3 as a facilitator of TGF-β-driven metastatic activity in cancer accents its potential as a target for therapeutic intervention to curb cancer spread and progression. Further, the review outlines the interaction of WAVE3 with the Abl non-receptor tyrosine kinase that is essential in coordinating cell movement and the positioning of focal adhesions and lamellipodia. Specifically, Abl kinase directly phosphorylates WAVE3, activating it to regulate actin polymerization, which in turn, influences the formation of lamellipodia and the dynamic assembly of focal adhesions. These focal adhesions serve as crucial points of contact between the cell and its external environment, facilitating signal transduction that informs cell direction and velocity. The precise modulation of these structures is integral to cell motility, impacting processes such as wound healing, immune cell migration, and cancer cell invasion.

WAVE3 was also identified as an important regulator in the progression of TNBC and treatment resistance. Although chronic exposure to chemotherapeutic agents does not lead to up-regulation of WAVE3 in breast cancer, WAVE3 expression is associated with chemoresistance through mechanisms involving the STAT1-HIF-1α-VEGF-A signalling axis (106). The study by Wang (107) illustrates the role of WAVE3 in stabilizing WAVE3-mediated stabilization of β-catenin leads to activation of the pathways of oncogenic signalling, that result in cell survival, and chemoresistance. WAVE3 likewise impacts the YB1-dependent activity of the transcription factor that triggers genes specifically expressed in cancer stem cell-like cell populations. The YB1 regulates the expression of β-catenin, thereby influencing the invasive capacity of triple-negative breast cancer (TNBC) tumours. Their findings further implicate that dual blocking of WAVE3 expression or phosphorylation, besides chemotherapy, will work in the direction of down-regulation of activity as well as expression of β-catenin, ultimately causing the inhibition of oncogenic behaviour of the chemoresistant TNBC cells. Moreover, recent findings by Mustafa Qasim et al. (108) revealed that reduced KISS1 expression – a known suppressor of metastasis – correlates with increased WASF3 (WAVE3) activity, suggesting a regulatory axis that promotes invasive phenotypes in TNBC cells. This was further supported by findings where the loss of KISS1 leads to up-regulation of ZEB1/2 and increased MMP9 activity, hallmarks of epithelial-to-mesenchymal transition (EMT) and extracellular matrix degradation. This study emphasizes the need to explore therapeutic strategies targeting the KISS1-WAVE3 signalling axis as a potential approach to mitigate TNBC progression.