Microtubule actin crosslinking factor 1 (MACF1) is a spectraplakin protein with a N-terminal actin-binding domain, plakin domain, an EF-hand calcium-binding domain, with a spectrin-repeat rod and C-terminal growth-arrest specific 2-related microtubule-binding domain. These structural domains enable MACF1 to perform its primary function as a crosslinker of microtubules and actin microfilaments (1-4), cytoskeletal filamentous proteins involved in vesicular trafficking, cytoarchitecture, cell division, and cell migration. In addition to its function as a cytoskeletal crosslinker, it is widely established that MACF1 plays a role in Wnt signaling, as a component of the Wnt signaling protein complex (axin1, beta-catenin and glycogen synthase kinase) that activates Wnt transmembrane proteins and subsequently induces Wnt transcriptional targets (5). Because several studies provide instructive and informative presentations of MACF1 structure and function (6-8), the focus in the present review is an overview of its role in the etiology of brain tumors, specifically glioblastomas.

Early seminal work by Goryunov et al (9) demonstrated that MACF1 has a significant and prominent role in the mammalian nervous system using in vivo tissue specific knockout technology. To that end, A Cre-loxP approach was employed to knockout MACF1 in the early stages of mouse nervous system development, which consequently compromised the organizational structure of the cerebral cortex and neuronal axon migration (9), while a more recent study by Ka et al (10), showed that MACF1 regulates GABAergic interneuron migration and positioning in the developing mouse brain using a conditional deletion approach. Several confirmatory studies, particularly in vitro knockdown and deletion experiments of MACF1, showed that the most important nervous system function of this spectraplakin protein is its crosslinking capacity during axon outgrowth and migration (11-15). Additionally, clinical nervous system manifestations of MACF1 alterations were previously reported in a study by Dobyns et al (16), which described missense variants and in-frame deletions within the growth-arrest specific 2-related microtubule-binding domain that resulted in brain malformations of children. Collectively, these studies corroborate a defined function of MACF1 during neuron outgrowth and axon migration as part of nervous system development (Fig. 1). However, despite robust data that MACF1 contributes to neuronal development and maturation, few investigations have examined its function in glial cells (astrocytes, oligodendrocytes and microglia) and non-neuronal cells that provide support and protection for neurons.

Several investigations have provided experimental evidence that show various MACF1 genetic abnormalities (Table I). One of the earliest studies implicating MACF1 in cancer was described in 2011; alternative splicing in adenocarcinoma patients was examined using microarray analyses and reverse transcription polymerase chain reaction (17). MACF1 was identified as one of four alternatively spliced transcripts that may contribute to non-small cell lung cancer (NSCLC) tumorigenesis as a consequence of exon alterations (17). In support of studies that evaluated MACF1 exon alterations as an underlying inducer of adenocarcinoma tumorigenesis, whole exome sequencing revealed MACF1 mutations in renal cell carcinomas and endometrial cancer as a genetic driver of tumorigenesis in these cancers (18,19). Furthermore, a recent study by Tian et al (2020), identified MACF1 mutations as a correlation of poor prognosis in patients with breast cancer. With respect to genetic alterations of MACF1 in brain tumors, specifically glioblastomas, cancer genome atlas cbioportal (https://www.cbioportal.org/) analyses revealed that 5% of patient samples consisted of mutations or amplifications (21-23).

MACF1 has been described to play a significant role in cancer development, primarily through its influence on cellular processes such as proliferation, migration and apoptosis. Specifically, in acute myeloid leukemia (AML), MACF1 overexpression was associated with poor overall survival and attributed to the promotion of AML cell proliferation by affecting pro-tumorigenic downstream targets, Runx2 and the PI3K/Akt signaling pathway (24). By contrast, silencing MACF1 in AML cells led to reduced proliferation and provided evidence of this spectraplakin as a therapeutic target for managing this type of leukemia (24). Consistent with these findings, MACF1 expression was also upregulated in serous ovarian cancer and correlated with shorter recurrence-free survival and overall survival (25), while in NSCLC cells, particularly in gefitinib-resistant cells, circ_MACF1 (a circular RNA form of MACF1) regulates drug sensitivity and cellular behavior through its interaction with miR-942-5p and TGFBR2(26). This axis influences cell proliferation, migration, and invasion while promoting apoptosis and sensitivity to gefitinib, suggesting that targeting circ_MACF1 could overcome resistance to EGFR inhibitors such as gefitinib in NSCLC. Collectively, these investigations provide a premise for the role of MACF1 in the etiology and progression of central nervous system glial-derived tumors. It is also noteworthy that previous investigations of plakin family members, plectin and desmoplakin, have been described as biomarkers for glioblastomas (27-29).

Malignant brain tumors in the central nervous system are arguably the deadliest types of cancers diagnosed, with glioblastomas being the most common, with an average median survival of 12-14 months and a five-year survival rate of ~5% (30,31). A major contributing factor to the poor prognosis of these cancers is their complex genetic heterogeneity that underlies their pathological origin, evolution and therapeutic resistance. Identification of novel mediators of disease transformation, progression and therapeutic evasion are critical to advancing strategies for the clinical management and treatment of these cancers. It is widely established that the evolution of glioblastomas from astrocytes and neural progenitor stem cells are a consequence of genetic mutations, deletions and amplifications in phosphatase and tensin homolog, neurofibromin1, p53, epidermal growth factor receptor and platelet-derived growth factor receptor (32-35). Genetic alterations in these oncogenes and tumor suppressors that contribute to glioblastoma initiation and progression are supported by knockout and genetically engineered mice models (36,37).

Although aberrant genetic abnormalities of receptor tyrosine kinases, phosphatases and transcription factors have been attributed to glioblastoma development, cytoskeletal proteins such as nestin, vimentin and alpha-actinin have also been identified as contributors to the inception of these tumors based largely on expression analyses (38-40). The best characterized of these, nestin, an intermediate filament expressed in neural progenitor cells, has long been recognized as a contributing oncogenic element in glioblastomas. Experimentally, nestin positive neural stem cells have been demonstrated to give rise to gliomas in murine models when transduced with EGFRvIII (39). Paralleling expression patterns of nestin and the previously mentioned cytoskeletal proteins, Afghani et al (41), also observed that MACF1 expression was absent in normal brain tissue and low-grade gliomas (oligodendrogliomas and medulloblastomas) but displayed significant expression in glioblastomas, which have high recurrence and mortality rates. These data suggested that MACF1 is a potential oncoprotein and therapeutic target in high-grade astrocyte derived gliomas.

Despite the observation that MACF1 was expressed at high levels in glioblastomas and that negatively regulating its function impaired glioblastoma cell proliferation and migration, the role of this spectraplakin protein as a tumorigenic driver in cancer and glioblastomas specifically, has not been investigated. However, a preliminary assessment of MACF1 tumor transformation properties in normal astrocytes, one of the two cell types, along with neural progenitor stem cells, considered the cellular origins of glioblastomas was conducted to evaluate whether MACF1 perpetuates tumorigenic characteristics. To that end, unpublished data of MACF1 overexpression studies performed in normal astrocytes, previously demonstrated to express low MACF1 protein levels, displayed significant increases in cell viability and anchorage independent growth (42-44), indicators of the transformed phenotype in normal astrocyte cells (Fig. 2). These cellular responses are consistent with the aforementioned oncogenic role of cytoskeletal nestin in glioblastoma formation.

In addition to primary tumor development, secondary glioblastomas and disease progression as manifested by tumor recurrence resulting from normal tissue invasion is a collateral oncogenic process, also derived from genetic abnormalities as aforementioned, that leads to poor disease management and high mortality rates. Further support of the pro-tumorigenic role of MACF1 in glioblastomas, specifically as it relates to disease recurrence, was also demonstrated in unpublished experimental studies (42-44), which showed that MACF1 overexpression increased astrocyte cell migration, a prerequisite cell behavior of metastatic invasion. Taken together, these cellular biological data (42-44) provide evidence that spectraplakin protein is causally involved in primary and secondary glioblastoma tumorigenesis and expands the notion that MACF1 contributes to tumor development due to mutations and alternative splicing events identified in endometrial cancer, renal cell carcinomas and lung cancers, respectively (17-19).

As previously discussed, early investigations have established that MACF1 plays a mechanistic role in Wnt-mediated signaling. Specifically, MACF1 downregulation was demonstrated to reduce nuclear β-catenin and transcriptional activation of Wnt responsive genes (5). More importantly, aberrant regulation of Wnt signaling is also known to contribute to tumor proliferation and migratory invasion in malignant brain tumors (45-48), providing a correlative association that the onco-tumorigenic impact of MACF1 is related to its interaction with the Wnt signaling pathway (Fig. 3), a well-characterized mechanistic mediator of tumor cell survival and proliferation. As it pertains to central nervous system-derived cancers such as glioblastomas, the most direct evidence for a mechanistic role of MACF1 intracellular signaling in these tumors was provided by studies from Afghani et al (41), which showed that downregulation of MACF1 reduced Axin and phospho-β-catenin protein levels in glioblastoma cells (41). Furthermore, studies by Bonner et al (49) in irradiated glioblastoma cells revealed that genetic silencing of MACF1 reduced the expression of ribosomal protein s6, a downstream effector target of mTORC1, and consequently sensitized these astrocyte-derived cancer cells to radiation (49). This is particularly significant given the established roles of both the Wnt signaling pathway and PI3K-Akt-mTOR signaling axis as contributors in glioblastoma progression (Fig. 3), invasion and therapeutic resistance (50-53).

More importantly, when MACF1 is genetically silenced, Wnt signaling mediators and mTOR effector proteins are functionally impaired (Fig. 3). Given the breadth of these pro-tumorigenic signaling pathways in several cancers and glioblastomas in particular, a number of investigations have examined small molecule inhibitors targeting these pathways in glioblastomas (54,55). Although signaling functions of MACF1 have been predominantly associated with positive regulation of the Wnt signaling pathway, additional studies in AML, a blood and bone marrow cancer have provided additional insights on intracellular signaling roles of MACF1. Specifically, silencing MACF1 function in AML cells was found to reduce runt-related transcription factor Runx2 expression and inactivated phosphatidyl inositol 3 kinase signaling (24). Furthermore, co-immunoprecipitation experiments in AML cells provided evidence that MACF1 interacts with leucine-rich repeat-containing protein 1(56), while osteogenesis studies revealed that MACF1 positively regulates the TCF4/miR-335-5p signaling pathway, consequently influencing bone formation (57). Collectively, these studies provide evidence that extend the mechanistic function of MACF1 beyond Wnt pathway.

Collectively, this suggests that MACF1 is a contributor to treatment resistance of glioblastomas by acting as a signaling mediator in divergent intracellular signaling cascades. However, despite the absence of MACF1 in normal human astrocytes and high expression levels in high-grade astrocytomas (19), as well as the onco-transformation properties of this spectraplakin protein in glial cells, small pharmacological inhibitory molecules targeting this cytoskeletal cross-linker have not yet been identified. Because MACF1 crosslinks microtubules and actin-filaments have prevalent biophysical roles in mitotic tumor cell division and migration, developing pharmacological agents that impair MACF1 provides a singular therapeutic target that disrupts tumor cell behaviors that lead to glioblastoma progression and therapeutic evasion.

Although the aforementioned experimental investigations provided evidence that genetic alterations of MACF1 were prevalent in several cancers, the role of MACF1 in cancer cell biology and as a neoplastic target had remained unexamined. To that end, studies by Afghani et al (41) and Bonner et al (49), were the first to investigate MACF1 as a cancer therapeutic target and demonstrated that inhibiting the functional expression of MACF1 alone and in combination with radiation and the clinically used DNA damaging agent, temozolomide, had antitumorigenic effects on glioblastomas (Table II), astrocyte-derived central nervous system tumors (57,58). Additionally, findings in glioblastomas along with those by Wang et al (58), revealed that negative regulation of MACF1 impaired glioblastoma cell migration and melanoma metastasis by decreasing the epithelial to mesenchymal transition (Table II) and thus provided evidence of the functional role of MACF1 in metastatic invasion (57,58).

To date, therapeutic agents that directly target MACF1 are not yet available. Given MACF1's role in cellular processes such as intracellular signaling and cell migration, which are often dysregulated in cancer, warrants the development and evaluation of anticancer drugs that target this cytoskeletal protein. Further rationale to support the feasibility of developing such drugs includes the role of MACF1 in cytoskeleton dynamics for maintaining cell shape, polarity and motility, which are important characteristics of cancer cell invasion and metastasis. It is also noteworthy that because of MACF1's role in Wnt signaling, which is often dysregulated in several cancers, inhibiting the function of this plakin protein represents a novel neoplastic target. However, a caveat to the druggability of MACF1 is its large size of ~600 kDa and the numerous structural domains that it contains. Additionally, engineering molecules that target such a large protein as well as bioavailability challenges posed by the blood brain barrier to access astrocytic glioblastomas, provide unique challenges.

The development of cancers are a consequence of combinatorial genetic factors and their expressed products that underlie intra- and inter-tumor heterogeneity. MACF1 is a potential novel tumorigenic protein that may contribute to the clinical etiology and progression of astrocyte-derived cancers such as glioblastomas that reside in the central nervous system, specifically the human brain, by perpetuating glial cell proliferation and invasion. The investigation of MACF1 in cancer biology, specifically glioblastomas, as a novel oncoprotein that contributes to the etiology and progression of these central nervous system-derived tumors warrants continued investigation. To further establish the pro-tumorigenic role of MACF1 in the evolution of brain tumors, it is essential to perform oncogenic analyses of MACF1 in more translational applicable model systems with diverse genetic backgrounds, such as orthotopic patient-derived xenograft brain tumor models. The utility of these model systems would provide further insight and perspective of MACF1 in the context of oncogenes that drive a plethora of intracellular signaling mechanisms and regulate tumorigenic cell behaviors such as the PI3K signaling pathway, one of the most prevalent in the perpetuation of tumorigenesis, and in the absence of tumor suppressors during oncogenic transformation. Additionally, pursuing the development of chemotherapeutic agents that target MACF1 will broaden clinical approaches beyond therapeutic agents such as vinca alkaloids that inhibit microtubules used to treat this disease.