Eg5 and Diseases: From the Well-Known Role in Cancer to the Less-Known Activity in Noncancerous Pathological Conditions

Eg5 is a protein encoded by KIF11 gene and is primarily involved in correct mitotic cell division. It is also involved in nonmitotic processes such as polypeptide synthesis, protein transport, and angiogenesis. The scientific literature sheds light on the ubiquitous functions of KIF11 and its involvement in the onset and progression of different pathologies. This review focuses attention on two main points: (1) the correlation between Eg5 and cancer and (2) the involvement of Eg5 in noncancerous conditions. Regarding the first point, several tumors revealed an overexpression of this kinesin, thus pushing to look for new Eg5 inhibitors for clinical practice. In addition, the evaluation of Eg5 expression represents a crucial step, as its overexpression could predict a poor prognosis for cancer patients. Referring to the second point, in specific pathological conditions, the reduced activity of Eg5 can be one of the causes of pathological onset. This is the case of Alzheimer's disease (AD), in which Aβ and Tau work as Eg5 inhibitors, or in acquired immune deficiency syndrome (AIDS), in which Tat-mediated Eg5 determines the loss of CD4+ T-lymphocytes. Reduced Eg5 activity, due to mutations of KIF11 gene, is also responsible for pathological conditions such as microcephaly with or without chorioretinopathy, lymphedema, or intellectual disability (MCLRI) and familial exudative vitreous retinopathy (FEVR). In conclusion, this review highlights the double impact that overexpression or loss of function of Eg5 could have in the onset and progression of different pathological situations. This emphasizes, on one hand, a possible role of Eg5 as a potential biomarker and new target in cancer and, on the other hand, the promotion of Eg5 expression/activity as a new therapeutic strategy in different noncancerous diseases.


Introduction
Kinesin Eg5, also known as Kinesin Spindle Protein (KSP) or KIF11 (from the gene that encodes Eg5), is a motor protein that belongs to the Kinesins superfamily (KIFs) 11.It is a motor protein associated with microtubules (MTs), as it uses the structure of MTs as a road and the hydrolysis of ATP into its motor domain as a source of energy to move along the microtubules [1].Eg5 is primarily identifed as a mitotic motor protein; in fact, its essential activity in the formation of bipolar spindles during mitotic cell division has been widely recognized.Between the prophase and prometaphase, the mitotic spindle MTs, starting from the two centrosomes, extend across the cell cytoplasm.Eg5 reaches maximum concentration during these steps; thanks to its homotetrameric structure, anchors itself to the plus end extremity of interpolar MTs, allowing its overlapping at the equator of the cell and, at the same time, the separation of the two centrosomes at the opposite poles of the cell [2,3].To allow bipolar spindle formation, diferent mechanisms of action coexist, such as phosphorylation or deacetylation of diferent Eg5 sites [4].Before entering mitosis, Eg5 is acetylated on lysine 890 in the tail domain and this form of Eg5 is inactive; to become active, a deacetylation is made by Histone Deacetylase 1 and a mitotic progression is possible [5].Ten, two additional phosphorylations on the tail domain of Eg5 should be required: the frst on the Tr-927, made by cyclin-dependent kinase 1 in prophase, allows the localization of Eg5 to centrosomal MT, where it accumulates.Ten, phosphorylation made by Nek6/Nek7, two serine-threonine protein kinases, can initiate centrosome separation and ultimately spindle bipolarity [6].Simultaneously, motor activity is regulated through phosphorylation of three tyrosine residues within the motor domain (Y211, Y125, and Y231) produced by kinases of the Src family (SFK) [7].All these mechanistic processes determine bipolar spindle pole formation, followed by correct mitotic cell division (Figure 1(a)).Perturbations in Eg5 functions or perturbations in the phosphorylation process of Eg5 activation determine the formation of a monopolar spindle, called a monoaster, with failure in mitotic cell division (Figure 1(b)) [3].Tis failure in the mitotic process and the formation of monopolar spindle, inevitably, trigger a programmed cell death.Te condition by which the cells with altered Eg5 functions are unable to conclude the mitotic event is called "mitotic catastrophe" and triggers the apoptotic cascade as preferential programmed cell death [8,9].
In addition to the mitotic function, during the last decade, nonmitotic functions, correlated with an interphase pool of Eg5 in the cells, were highlighted (Figure 2).
In 2011, Bartoli et al. discovered that Eg5 is required for physiological levels of protein synthesis.It is likely that this kinesin is connected to ribosomes during the interphase and its activity is essential during the postinitiation phase of polypeptide synthesis (elongation/termination) by linking 80S ribosomes subunits to MTs during translation.More specifcally, Eg5 serves as a motile link between the ribosome and MTs and its in vitro inhibition is known to determine errors in the elongation and termination steps.How Eg5 binds to the ribosome is still unclear: a direct binding of Eg5 to the ribosome (Eg5 as a direct link between 80S of the ribosome and MTs) or an indirect binding (through an undefned linker molecule) are hypothesized [10].In addition to the role in polypeptide synthesis, another nonmitotic function identifed for Eg5 is in protein transport from the trans-Golgi complex to the cell surface: Eg5 interacts with the protein carrier, namely CARTS, and moves proteins, through CARTS, from the Golgi to the cell surface.Eg5 pharmacological inhibition determined an interruption of the activity of the CARTS carrier [11].Eg5 is also extensively studied as a critical protein that regulates angiogenesis, axonal branching, and cell motility, particularly in cancer models [12][13][14].However, it is unclear how Eg5 regulates these events.With respect to the relationship between Eg5 and angiogenesis, it has been elucidated that Eg5 is necessary for normal and abnormal vascular development.An upregulation of Eg5 was found in blood endothelial cells and lymphoblasts, as after angiogenic stimulation of chick embryos with VEGF-A, and also in tumor blood vessels.In vitro pharmacological inhibition of Eg5 decreases endothelial cell proliferation and migration, while in in vivo zebrafsh and chick embryos models, interference with Eg5 function causes developmental and vascular defects, along to inhibition of angiogenesis in tumor models [13].At the same time, Eg5 infuences the migration of diferent types of cells, such as neurons and tumor cells.In neurons, Falnikar et al. showed an Eg5 regulatory potential for the migration process and the interruption of migration when the neuron reaches its destination [15].In tumor cells, such as human pancreatic cells, pharmacological inhibition of Eg5 decreases cell migration and invasion [12].
Te highest number of published articles on Eg5 is related to the role of this kinesin in cancer.High Eg5 expression levels have been highlighted for decades in diferent tumors, with a poor prognosis for patients and pharmacological Eg5 inhibitors were tested as a new therapeutic strategy.However, this protein plays an important role in other less well-known diseases, for which a stimulation of Eg5 expression could be a valid therapeutic approach, even if, compared to the high amount of tested Eg5 inhibitors for cancer treatment, Eg5 activators are currently not available.
Te aim of the present review (Figure 3) is, on the one hand, to summarize recent fndings on the correlation between Eg5 and the onset and progression of cancerous malignancies, and, on the other hand, to shed light and summarize old and recent fndings on the less known correlation between Eg5 and specifc diseases, such as Alzheimer's disease (AD), the genetic form of microcephaly typically associated with retinopathy, genetic retinopathy conditions, and others.

Eg5 in Tumor Onset and Progression
Te correlation between Eg5 expression and cancer origin and progression has been well known for decades and is related to an unfavorable prognosis [16][17][18].Indeed, Eg5 expression levels in nontumor proliferative tissues are signifcantly lower than those recorded in tumor tissues, allowing to consider this protein as a possible prognostic biomarker and a therapeutic target.Nowadays, the scientifc community is still focused on searching for Eg5 inhibitors able to counteract tumor onset and progression.Several recent reviews focused attention on the role of Eg5 inhibitors as a cancer therapeutic strategy, highlighting new fndings on this topic [19][20][21][22][23].As all recent reviews on Eg5 and cancer are mainly focused on the inhibition of this protein and the discovery of new drugs, in this section, the current literature, from 2021 to 2023, is discussed on the correlation between Eg5 and diferent cancerous malignancies (resumed in Table 1), from a biological point of view.
Te literature of the last 2 years shed light on lung and gastro-intestinal tract carcinomas as tumors with the highest correlation with Eg5 expression, followed by reproductive system carcinomas (breast, ovarian, and prostate) and central nervous system tumors; in other tumors (such as bladder cancer, renal cancer, and thyroid cancer) Eg5 overexpression is known, but less papers are available.Based on the abundance of literature information regarding Eg5 and these tumors, we started our discussion with those tumors for which more information is available, followed by tumors with a less known correlation with Eg5 (Figure 4).

Lung Carcinoma.
Lung cancer is one of the most common tumors worldwide.In 2020, it was the second, after female breast cancer, for the number of new cases (11.7%) and, despite the available therapeutic approaches, it remained the leading cause of cancer mortality (18%) [67].Non-small-cell lung carcinoma (NSCLC) is the most widespread and common type of lung cancer and represents more than 80% of the total number of lung cancers, with lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) being the most common subclasses [68].Te

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Biochemistry Research International necessity of new targets to hit is undoubtedly indispensable to improve the life expectancy of people.A consistent number of fndings between 2021 and 2023 have identifed Eg5 as a prognostic factor and a possible new therapeutic target for patients afected by lung cancer.An integrated bioinformatic data set analysis of 497 LUAD tissues and 54 normal tissues from the Cancer Genome Atlas (TCGA) highlighted KIF11 as a pivotal gene from the ten genes identifed with the highest expression levels.KIF11 was highly expressed in LUAD compared to normal tissues and its high expression is correlated with poor overall survival and worse progression-free survival, in addition to the positive association with tumor grade.Furthermore, KIF11 expression appears to be related to the formation of immune infltrating cells in the tumor microenvironment: Resting NK cells and memory CD 4+ T cells, regulatory T cells, and monocytes were signifcantly associated with KIF11 expression and with overall survival of patients with LUAD.KIF11 knockdown determined an inhibition of proliferation, migration, and invasion of lung adenocarcinoma cells A549 and PC-9, in addition to arrest of the G2/M phase cell cycle and induction of apoptosis [24].How KIF11 modulates the onset and progression of LUAD is not yet known.Recent interesting information is the correlation Biochemistry Research International found between noncoding RNAs (nc-RNAs) and KIF11.nc-RNAs are small RNAs with no ability in protein coding, based on their length, shape, and location, and are classifed in three main nc-RNAs: circ-RNA, miRNA, and long noncoding RNAs (lnc-RNA).lnc-RNAs and circ-RNAs can control gene expression by modulating transcription factors, regulating protein-protein interactions, and epigenetic modulation of chromatin; all these events play roles in tumor-related processes, such as proliferation, invasion, and metastasis [69].What was discovered in recent years is that the progression of LUAD could be triggered and improved via the VPS9D1-AS1/miRNA-30a-5p/KIF11 axis.VPS9D1-AS1 is an lnc-RNA that, through interactions with specifc miRNA, in particular by sponging diferent miRNA, promotes malignant tumor progression [70,71].In LUAD, VPS9D1-AS1 results overexpressed and appear to facilitate proliferation, migration, and invasion of LUAD cells.In this malignancy, VPS9D1-AS1 sponges a specifc miRNA, miRNA-30a-5p, an endogenous noncoding RNA that normally suppresses the progression of diferent tumor types [72][73][74] and whose expression is downregulated in LUAD.KIF11 is a downstream gene of miRNA-30a-5p; the malignant phenotype of LUAD is inhibited by the ability of miRNA-30a-5p to target and inhibit KIF11 overexpression.Te possibility that VPS9D1-AS1 targets KIF11, through sponge activity in miRNA-30a-5p, promoting malignant progression of LUAD, could be a new possible axis underling LUAD onset and progression (Figure 5) [26,27].At the same time, Balasundaram et al. focused their attention on the regulatory axis of circRNA-miRNA-mRNA in NSCLC.Tey identifed 12 dysregulated pivotal genes associated with NSCLC, including KIF11.In particular, they analyzed the correlation between circ-RNA-miRNA-mRNA with the identifed hub genes.Tese 12 genes encode 12 mRNAs which, in turn, are linked to 8 miRNAs associated with 1 circRNA.Regarding KIF11, they found that a specifc circ-RNA (circ_0003812) is associated with miRNA-200b, which in turn is related to KIF11.Tis axis could infuence the biological processes of NSCLC and could represent a new blockable biomarker axis [30].Trough the Kaplan-Meier survival analysis, KIF11 was also identifed among the 9 hub genes related to poor overall survival in patients with NSCLC, again with high expression in tumor tissues compared to nontumor tissues [25].A microarray data set from the Gene Expression Omnibus (GEO) database identifed other 9 genes correlated with the progression of NSCLC: in the list KIF11 appears as an upregulated gene involved in tumorigenesis, progression, and resistance to cisplatin, its knockdown decreased the proliferation of A549 and SPCA1 cells [28].KIF11 was also identifed as a key gene involved in the tumorigenesis of benzo(a)pyrenediolinduced NSCLC [29].
Based on these fndings reported in the last 2 years, it is possible to include Eg5 between potential biomarker for lung cancer.In LUAD diagnosis, diferent biomarkers are currently available: KRAS is the most frequent mutated gene with around 30% of mutation frequency, followed by EGFR (around 10% of frequency in mutation), MET (8% of mutation frequency) and ALK (4-7% of rearrangement frequency).Other biomarkers show mutations or rearrangements frequency ranging from 1% to 8%, such as ROS1, NTRK1, HER2, BRAF, DDR2, and PD-L1.In NSCLC, less biomarkers are available: FGFR shows 20% of amplifcation frequency, followed by 16% of PTEN gene deletion and 7% of PIK3CA gene mutation, while KRAS, EGFR, and MET show approximately 5% of mutations frequency [75].To obtain this panel of biomarkers could represent a crucial step to fne tune a tailored target therapy.For instance, patients with LUAD KRAS G12C mutated are treated with KRAS inhibitors, while patients with PD-L1/PD1 are eligible for immunotherapy.However, for most of mentioned biomarkers, the mutation percentage is very low and, resistance processes are often responsible for low response to therapy.In addition, for NSCLC, less biomarkers are available compared to LUAD.Eg5 could become a new valid target to enlarge the panel of available biomarkers.Furthermore, focusing on this point, Eg5 expression can be deeply investigated between LUAD and NSCLC thus allowing to distinguish between the two tumor types.Lung cancer KIF11 serves as an independent prognostic factor and therapeutic target for patients with lung adenocarcinoma Li et al. [24] 2021 Identifcation and integrate analysis of key biomarkers for diagnosis and prognosis of non-small-cell lung cancer based on bioinformatics analysis Gong et al. [25] 2021 Characterization of cell cycle-related competing endogenous RNAs using robust rank aggregation as prognostic biomarker in lung adenocarcinoma Yang et al. [26] 2022 LncRNA VPS9D1-AS1 promotes malignant progression of lung adenocarcinoma by targeting miRNA-30a-5p/KIF11 axis Liu et al. [27] 2022 Identifcation and verifcation of hub genes associated with the progression of non-small-cell lung cancer by integrated analysis Mengyan et al. [28] 2022 KIF11, a plus end-directed kinesin, as a key gene in benzo(a) pyrene-induced non-small-cell lung cancer Ling et al. [29] 2022 In silico analysis revealed the potential circRNA-miRNA-mRNA regulative network of non-small-cell lung cancer (NSCLC) Balasundaram et al. [30] 2023 Colon cancer KIF11 is upregulated in colorectal cancer and silencing of it impairs tumor growth and sensitizes colorectal cancer cells to oxaliplatin via p53/GSK3β signaling Zhou et al. [31] 2021 Prognostic impact and functional annotations of KIF11 and KIF14 expression in patients with colorectal cancer Neska-Długosz et al. [32] 2021 Overexpression of the NEK9-EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer Kim et al. [33] 2023

Gastric cancer Identifcation of the hub genes and prognostic indicators of gastric cancer and correlation of indicators with tumor-infltrating immune cell levels
Ji et al. [34] 2021 Bayesian hierarchical lasso cox model: A 9-gene prognostic signature for overall survival in gastric cancer in an Asian population Chu et al. [35] 2022 Synthesis of new 2-aminothiazolyl/benzothiazolyl-based 3,4-dihydropyrimidinones and evaluation of their efects on adenocarcinoma gastric cell migration Sagha et al. [36] 2022 Negative modulation of the angiogenic cascade induced by allosteric kinesin Eg5 inhibitors in a gastric adenocarcinoma in vitro model Ricci et al. [37] 2022 Hepatocellular carcinoma Identifcation of hub genes and their correlation with immune infltration cells in hepatocellular carcinoma based on GEO and TCGA databases

Gastrointestinal Tract and Tumors of the Annexed Organs.
Gastrointestinal tumors, along with tumors of the annexed organs, are among the most diagnosed tumors worldwide.At the frst position there is colon cancer, considered in the Global Cancer Statistic 2020 the ffth cancerous malignancy among the 36 common cancers, with new cases incidence of 6%, followed by stomach, liver, and rectum cancers.Pancreas cancer is less diagnosed, with 2.6% of new cases of incidence but 4.7% of mortality rate [67].Surprisingly, based on the collected literature of the last two years and half, Eg5 appears to be a common altered marker in diferent gastrointestinal tumors.Bioinformatic analysis of the TCGA data set revealed 29 upregulated kinesins in colon cancer, with strong overexpression of KIF11 in cancerous colon tissues compared to normal tissues.Te higher expression of KIF11 is also related  Biochemistry Research International to the higher grades of tumor clinical stage T, M, and TNM, but not with stage N.A substantial reduction in in vivo and in vitro tumor growth is recorded after KIF11 knockdown, and, interestingly, silencing of KIF11 increased the sensitivity of colon cancer cells to oxaliplatin, a common anticancer drug used in colon cancer treatment; in fact, a reduction in the IC 50 value of oxaliplatin and colony formation was recorded.Tis efect could be due to an aberrant activation of p53 pathway and a deactivation of GSK3β signaling, both efects triggered by KIF11 removal [31].KIF11, together with KIF14, another kinesin involved in the correct cell division, could be responsible for the pathological genomic instability of colon cancer, and their levels refect the clinical outcome.Both these kinesins could be used to stratify patients with better and worse overall survival: overall survival is negative when high KIF11 protein levels, high KIF11 protein together with low KIF14 protein levels, or low KIF11 and KIF14 mRNA levels, were found [32].Eg5 is also a potential metastatic marker in T3 stage colon cancer, together with NEK9.Te proteins of NEK family are cell cycle dependent proteins; since years it is well known that polo-like kinase 1, in collaboration with cyclin-dependent kinase 1, early phosphorylates NEK9 in mitosis, which in turn activates the NEK6/NEK7, members of the NEK family that phosphorylate Eg5 at S1033 in its tail domain, allowing its motor function and establishing a bipolar spindle [76,77].An overexpression of NEK9-Eg5 is recorded in patients with colon adenocarcinoma along with a distant metastasis association, thus representing a new possible biomarker to predict the metastatic potential in patients with T3 colon cancer [33].
Te new prognostic model, based on a gene signature developed by Chu and co-workers, shows a 9-gene signature to predict the overall survival of patients with gastric cancer in Asian population: among 9 identifed genes, KIF11 emerged [35] and these data could be extended to other populations.In fact, in a previous bioinformatic analysis, carried out by Ji et al., through the TCGA gastric adenocarcinoma dataset, 10 main genes of gastric cancer were highlighted, among which KIF11 is included [34], thus explaining why researchers are still focused on the research of the best Eg5 inhibitor to treat gastric cancer [36].Moreover, an important role for Eg5 is also revealed in controlling in vitro nonmitotic processes directly related to the aggressiveness of gastric cancer, such as angiogenic and migratory events.Indeed, efcient pharmacological inhibition of Eg5 determined a substantial reduction of AGS gastric adenocarcinoma cells and a negative modulation of the angiogenic event, presumably through the involvement of PI3K-Akt-VEGF and Erk-VEGF pathways [37].At the same time, this pharmacological inhibition acts on Eg5 mitotic functions, obtaining a valid reduction in AGS proliferation, with monoaster formation [78].
Hepatocellular carcinoma (HCC) is one of the cancer forms possessing the highest correlation with KIF11 overexpression.In the diagnosis and screening of HCC, serum alpha-fetoprotein is commonly used as molecular marker [79], however it is urgent to fnd new possible upregulated markers.14 overexpressed hub genes were identifed through an extensive and detailed bioinformatic algorithm and KIF11 is found among them.In this tumor model, upregulation of these genes is related to a worse overall survival time of patients.Interestingly, as previously reported for LUAD, there is a thin, but important correlation between KIF11 overexpression and tumor infltrating immune cells: CD 4+ memory activated T cells, macrophage M0, T cell follicular helper, regulatory T cells have a signifcant positive correlation with KIF11 overexpression [38].Te bioinformatic results were confrmed using 108 samples of surgical resection of HCC: Eg5 mRNA expression levels allowed to divide patients' samples into three tertile groups with high, medium and low Eg5 expression levels.Te results showed that overall survival and disease-free survival of patients with low levels of Eg5 mRNA expression are better compared to those with medium and high levels of Eg5, allowing to assume that high Eg5 expression could be associated with a poor prognosis of HCC [39].Hu et al. confrmed that KIF11 is negatively correlated with overall survival and disease-free survival of HCC patients, and its expression is positively correlated with tumor size: in vitro cell proliferation and tumor growth are signifcantly inhibited after KIF11 knockdown [40].Te molecular mechanism underlying Eg5 and HCC is unclear and further research is required to elucidate it.Zheng and co-workers hypothesized a regulation of Eg5 expression controlled by P21-activated kinases 6 (PAK6), a regulatory protein of cell migration, cell-cell adhesion, and apoptosis.When PAK6 is depleted in in vitro and in vivo models of HCC, overexpression of Eg5 is found, with a resulting formation of a multipolar spindle and cell cycle progression.In contrast, a knockdown of Eg5 completely inverted cell cycle progression and multipolar spindle formation together with PAK6 overexpression are highlighted.Tese results revealed that the outcome of patients with HCC could be infuenced by the interaction between PAK6 and Eg5, promoting tumor progression [41].As previously reported for LUAD, lnc-RNAs could play a decisive role in the correlation between Eg5 expression and HCC: one lncRNA, namely SNHG1, is correlated with in vitro expression of 6 upregulated hub genes connected with cell cycle progression, including KIF11.Knockdown of SNHG1 decreased the expression of these genes, along with the viability of two HCC cell lines, and determined a phase G1 cell cycle arrest acting as competitive endogenous RNA.Tis study emphasizes another possible pathway involved in KIF11 in HCC [43].Lastly, Cao and coworkers found that KIF11 overexpression in HCC could also be related to epigenetic modifcations, such as DNA methylation; in fact, among the genes exhibiting hypomethylation KIF11 is included, confrming once again the strong relationship between Eg5 expression and HCC and shedding light on another possible mechanism able to explain this correlation [42].
Based on these evidences, HCC is one of the main tumors with high expression of Eg5.A wide range of biomarkers are available for HCC diagnosis, from protein antigens (such as DKK-1, GP73, EMA, OPN, and others), to enzymes and isoenzymes (DCP, GGT, AFU, and PON1) [80].Eg5 could be a new valid biomarker to be added in the 8 Biochemistry Research International panel of protein markers and to be analyzed by immunohistochemistry after tumor tissue biopsy: high rates of Eg5 expression could modify the therapeutic approach, with the possibility to add an Eg5 inhibitor to the standard therapy.In addition, future researches could detect possible association between Eg5 expression levels and tumor stage or identify Eg5 as a potential metastatic marker: transcriptome analysis could be a valid approach for this type of identifcation [81].Pancreatic adenocarcinoma (PAC) is the second tumor of the gastrointestinal tract for which literature in the last 2 years demonstrated that a link between Eg5 expression and tumor onset exists.Bioinformatic analysis revealed diferentially expressed genes, among which KIF11 is included, correlated with tumorigenesis and PAC development, and confrming these assumptions in in vitro models of pancreatic ductal adenocarcinoma cell lines.Overexpression of KIF11 is related to a worse overall survival of PAC [46].Gu and colleagues highlighted a link between KIF11 and the mevalonate (MVA) metabolic pathway: recent fndings revealed a causal association between cholesterol intake and PAC, meaning that the risks of PAC are closely dependent on cholesterol metabolism.Tis research group showed an interaction between Eg5 and SREBP2, the main regulator of MVA: it seems that high expression of KIF11 increased the expression of the SREBP2 protein and reduced its ubiquitinmediated degradation.Promotion of cell growth, migration, stemming and colony formation, mediated by KIF11, depending on SREBP2, is highlighted: in vivo analysis revealed that PDA growth with high expression of KIF11 is controlled by targeting MVA biogenesis.Collectively, these fndings suggest that KIF11 could activate MVA cross-talk to promote PAC [45].As in colon cancer, also in PAC the expression of KIF11 and/or KIF14 could be identifed as a discrimination marker between patients with better and worse overall survival, independently of other relevant clinical risk factors [44].

Tumors of the Male and Female Reproductive Systems.
According to collected literature from 2021 and 2023, female reproductive system tumors having the highest correlation with Eg5 are breast cancer and ovarian cancer, while for male reproductive system tumors, a slight association was found between Eg5 and prostatic cancer.
Breast cancer (BC), with 11.7% incidence and 6.9% mortality in 2020, represents the frst widespread cancer among women [67] and the need for a new target to treat this malignancy stimulates researchers to fnd new possible diagnostic and prognostic biomarkers.
Trough a bioinformatic analysis, diferentially expressed genes correlated with cell division, cell proliferation, and BC signal transduction were identifed.Among them, KIF11 emerged as associated with poor overall survival of patients [49,52].A possible signaling pathway that promotes BC proliferation through Eg5 activity is the TRFA4/Eg5 axis.TRFA4, acronym for Tumor Necrosis Factor Receptor Associated Factor 4, is an E3 ubiquitin ligase regulating the ubiquitination of diferent proteins.Both TRFA4 and Eg5 are overexpressed in BC and a positive correlation between them was found.Eg5 seems to interact with TRFA4 in the cytoplasm of BC cells, where the latter blocks the ubiquitination of Eg5 through an inhibition of Smarf2 (a promotor of Eg5 ubiquitination) activity.TRFA4 blocks the interaction of Smarf2/Eg5 inhibiting Eg5 ubiquitination catalyzed by Smurf2 and upregulating Eg5 levels, thus increasing and stabilizing Eg5 levels during mitosis (Figure 6).With this mechanism, BC proliferation is promoted and apoptosis inhibited [82].
KIF11 is also one of the 10 hub genes identifed having a high and signifcant interaction with a BC marker, the thyroid hormone receptor interactor 13 (TRIP13), a protein acting in the spindle assembly checkpoint.Te proteinprotein interaction (PPI) network revealed that KIF11 could be a deregulator of TRIP13, representing a second possible therapeutic target [50].
Eg5 role in BC is also related to the molecular and histological classifcation of BC according to which three diferent types of BC can be recognized: BC positive for estrogen receptors (ER) and progesterone receptors (PR) (ER + , PR + ), BC positive for ER, PR, and for human epidermal receptor 2 (HER2 + ), BC positive only for HER2 and triple negative BC (TNBC, negative for ER, PR, and HER2 receptors) [83].KIF11 could be a target in TNBC, where its pharmacological inhibition revealed a slowdown in tumorigenesis and cancer progression in in vivo xenograft models.Interestingly, this pharmacological inhibition determined not only a downregulation of KIF11 expression, but also a downregulation of Aldehyde Dehydrogenase 1 family member A1 (ALDH1-A1): this is a marker of cancer stem cells (CSCs) and its high expression is correlated with poor overall survival [48].A previous study demonstrated that endogenous administration of KIF11 promoted the self-renewal of BC cells through BC stem cells and that after silencing of KIF11 a decrease in CSC markers is highlighted, ALDH1 included [84]; as a consequence, it is possible to state that a positive correlation between KIF11 expression and cancer stem markers exists, thus representing a possible therapeutic target.Eg5 could also be a valid marker in ER + /PR + BC: again, its pharmacological inhibition demonstrated a reduction in cell viability and proliferation probably correlated with the induction of apoptosis when Eg5 is completely inhibited: In parallel, a role for Eg5 in controlling events not related to mitotic activity, such as migration, invasion, and the occurrence of angiogenesis, was revealed.Indeed, after efcient pharmacological inhibition of Eg5 in an in vitro ER + /PR + BC model, a slight inhibition of cell invasion was highlighted and a higher migration inhibition was reported, probably through a modulation of the MMP-9/NF-kB pathway.Te transcription factor NF-kB, through a translocation into the nucleus, activates the transcription and protein secretion of the MMP-9 gene, which in turn is responsible for the degradation of the extracellular matrix, allowing cancer cells spreading.In ER + /PR + BC model, represented by MCF7 cells, inhibition of Eg5 determined a downregulation of MMP-9 protein expression levels that refects NF-kB protein levels, thus supposing a key role for Biochemistry Research International Eg5/MMP-9/NF-kB axis in controlling cell migration.At the same time, a reduction in HIF-1 -/VEGF protein expression levels was highlighted [51].
Considering the relevance of Eg5 in BC and the hypothetic mechanisms of action used by this protein to induce the onset or progression of tumor, it could be a new possible biomarker in addition to all the panel of markers already known: ER, PR, HER2, p5, Ki-67, BRCA1/BRCA2, PTEN, and others [85].In particular, considering the variability of this tumor and the resistance to the current standard of care, the necessity to identify new biomarkers is still urgent [86]; Eg5 emerged as a new possible target, and it could be revolutionary for patients' treatment.
Using four microarray datasets, Zhao and colleagues investigated common diferentially expressed genes in ovarian cancer.Te KIF11 gene was identifed among 20 genes highlighted through the PPI network and among the 6 genes whose overexpression is related to worse overall survival and progression-free survival in patients with ovarian cancer, together with a second kinesin called KIF23.Te bioinformatic obtained results have been validated by an in vitro ovarian cancer model, represented by SKOV3 cell line: silencing of KIF11 results in a signifcant reduction of cell proliferation probably due to induction of apoptosis, as demonstrated by the increased activation of Caspase 3/7.Furthermore, as previously reported for other tumor models, KIF11 knockdown determines the efect not only on mitotic activity, but also on nonmitotic activity: siKIF11 reduced the invasion of SKOV3 cells and modifed the expression levels of epithelial to mesenchymal transition genes, such as an upregulation of E-cadherin mRNA expression and a downregulation of N-cadherin and vimentin mRNA levels [54].Te bioinformatic results were also confrmed by Dong et al. [53], suggesting that further investigation is required to better clarify Eg5 role in this malignancy.
Regarding tumors of male reproductive system, prostatic cancer (PC) is the third for the number of new cases (7.3% incidence), after breast and lung cancer, in the Global Cancer Statistics 2020 [67].Recent bioinformatic analysis of TCGA datasets, including RNA-Seq data from prostate adenocarcinoma, allowed the identifcation of KIF11 among genes having the highest signifcant correlation with PC.PC cell diferentiation is determined by using the Gleason score, a number ranging from 2 to 10 identifying how many prostatic cells in tumor tissue are diferent from normal tissue, with the objective of establishing the aggressiveness of the tumor: values of 8 or higher correspond to a poorly diferentiated tumor and are associated with a poor prognosis [87].KIF11 gene expression was previously shown to be increased in PC samples having a Gleason score of 8 compared to samples with a Gleason score of 7 (moderately diferentiated tumors) [88].Pudova and colleagues found a strong negative correlation between KIF11 expression and tumor progression-free survival, thus confrming the fact that an Eg5 expression leads to worse PC prognosis for patients compared to those with lower levels of Eg5 [55].Additionally, among the seven central genes, KIF11 could also represent a marker of bone PC metastasis: in fact, it was found a positive correlation between KIF11 and VEGF, both associated to poor metastasis-free survival (as a surrogate for overall survival).KIF11 and VEGF are together associated with higher T stage, with prostate specifc antigen level, and with Gleason score [56].

Central Nervous System (CNS) Tumors.
Eg5 is also upregulated in diferent CNS tumors, with a great extent for gliomas and the highest grade of glioma, glioblastoma (GBM), thus shedding light on the possibility of using this kinesin as a new possible target.Glioma data obtained with the TCGA database revealed 14-18 hub genes correlated with the worse survival prognosis and among them KIF11 is included [57,62].Upregulation of Eg5 is not only found in well-diferentiated glioma cells, but also in more aggressive glioma stem cells (GSCs), as previously also revealed for other types of cancer.Tis subpopulation of cells with stemness and self-renewal characteristics, particularly aggressive in GBM, has the ability to escape from radiation and chemotherapy, constantly regenerating the tumor mass [89].Te expression of Eg5 is closely related to GSC markers [14].Indeed, Liu and co-workers demonstrated that Eg5 10 Biochemistry Research International promotes the expression of GSC markers, such as KLF4, OCT4, or NANOG, and the expression of the CD133 stemness marker, thus promoting the stemness associated with chemoresistance in TP53 mutant GBM.In addition, they found that Eg5 is associated with the expression of diferent cyclins, such as CDK1: the overexpression of Eg5 in the GBM model increased the expression of cyclins, modulating cell cycle progression [58].Recently, Kenchappa and colleagues found a possible resistance mechanism developed in GBM to the administration of a well-known Eg5 inhibitor, ispinesib.Tey discovered a suppression of ispinesib apoptosis induction through double phosphorylation of STAT3.
In GBM, STAT3 is a transcriptional regulator of the mesenchymal phenotype [90] which inhibits the induction of apoptosis and drives the self-renewal of cells, allowing chemoresistance [91,92].Two main phosphorylations are involved in the induction of resistance to ispinesib: the frst phosphorylation interests the Y705 residue, performed by SCR proteins that inhibit STAT3-mediated transcription of genes related to antiapoptotic proteins.Te second phosphorylation is on the S727 residue of STAT3, performed by the epidermal growth factor receptor (EGFR) which suppresses STAT3-mediated mitochondrial apoptosis duction.Te resistance of GBM to Eg5 inhibitors could be due to the aforementioned phosphorylations: only one of the two phosphorylations is sufcient to induce a resistance mechanism to kinesin spindle protein inhibitors.A combined inhibition of SRC and EGFR with Eg5 inhibition could reverse this resistance process [59].At the same time, targeting Eg5 could represent an approach to overcome GBM resistance to current standard of care: e.g., bevacizumab is an anti-VEGF antibody currently used in recurrent GBM, although resistance phenomena are responsible for a reduction of efcacy.Among the key regulators of resistance to bevacizumab, phosphofructokinase-1 muscle isoform (PFKM), emerged with Eg5 is an essential partner to trigger this molecular phenomenon: cytosolic PFKM interacts with Eg5, which, in turn, promotes GBM invasion [61].Lastly, pharmacological inhibition of Eg5 in head and neck squamous cell carcinoma determined a blockage of cell proliferation by inducing a G2 cell cycle arrest and accumulation of cyclin B1 [60].

Others.
Collected literature referring to 2021-2023 period reports other cancerous malignancies associated with Eg5.In bladder cancer, the bioinformatic analysis screened revealed 55 genes upregulated and 86 downregulated.KIF11 is one of the most signifcant biomarkers among all and it could be a promising new prognostic biomarker [63].In cutaneous squamous cell carcinoma, immunohistochemical analyses highlighted a 70% of diferences in Eg5 expression compared to normal skin, disclosing a high expression of this kinesin [64].Immunohistochemical tissue analyses of 90 patients afected by clear cell renal cell carcinoma, followed for 7 years, revealed a high expression of KIF11 in the cytoplasmic fraction compared to the nuclear fraction, and this was positively correlated with tumor grade and mortality [65].Lastly, Eg5 appears to have developed into a diagnostic and prognostic tool for thyroid carcinoma: patients with the highest KIF11 levels had the worst clinical pathological features (T stage and intraglandular dissemination).KIF11 inhibition suppressed thyroid cancer cell proliferation, triggering the apoptotic pathway.Furthermore, KIF11also appeared to help the development of thyroid cancer tumors in mice.

Eg5 in Noncancerous Diseases
As previously extensively reported, the mitotic and nonmitotic functions of Eg5 and their relative inhibition are usually associated with cancer.However, Eg5 could also play a role in other types of diseases.Te main associations found in the literature are related to Eg5 expression and AD, microcephaly conditions (in particular, a specifc condition characterized by microcephaly with or without chorioretinopathy, lymphedema, or intellectual disability, MCLID), and retinopathies (including familial exudative vitreous retinopathy, FEVR), even if additional pathological situations are reported in which Eg5 emerged.In this second part of the review, the authors will analyze the literature on less common topics.

Alzheimer's Disease.
AD is a neurodegenerative disease for which specifc causes have not yet been elucidated.Tere is a genetic form of AD, defned autosomal dominant AD (ADAD) or sporadic AD (SAD).Classic symptoms of AD start when the two main lesions in the CNS (the accumulation of beta-amyloid peptides (Aβ), with the formation of amyloid plaques [93] followed by the hyperphosphorylation of Tau protein, which determines the formation and accumulation of neurofbrillary tangles (NTF) [94]) afect neurons involved in memory, cognition, and neurogenesis.Lesions alter the normal structure and function of the CNS.
Despite the extensive research focused on this topic, today pharmacological approaches are aimed at treating the symptoms' onset; however, improving early disease identifcation in the preclinical stage is a current challenge for researchers [95].
In contrast to what is reported about Eg5 and tumor onset and progression, in this type of disease the correct expression and function of Eg5 is essential to recover the AD defcit.Indeed, under AD conditions, cell cycle defects have been demonstrated [96]: almost 30% of aneuploid cells (in the CNS and peripheral tissues) are evident and premature centrosome division and chromosome mis-segregation are increased [97,98].In particular, an accumulation of Aβ, its precursor (the membrane-traversing amyloid precursor protein, APP), and enzymes normally cleaving the APP (the β-amyloid cleaving enzyme, BACE, and the presenilin (PS)containing c-secretase enzyme complex) were found in the mitotic spindle, especially at the poles of the spindle and kinetochores of neurons, and they appear particularly prone to degeneration.Te mechanism by which the mitotic spindle is afected in AD, inducing neuronal degeneration, was discovered by Borysov et al. [99].By using primary human somatic cells and cell-free Xenopus egg extract, they Biochemistry Research International found that Aβ accumulation impairs mitotic spindle through an inhibition of some mitotic kinesins, in particular Eg5, KIF4A, and MCAK.Te mechanism by which Aβ afects mitotic kinesin activity is diferent: MCAK is inhibited by a noncompetitive mechanism (direct inhibition of its activity), while inhibition of Eg5 and KIF4A occurs through a competitive mechanism, as Aβ interferes with the ability of both kinesins to interact with ATP and microtubules (Figure 7).Te result is the formation of over 30% of aneuploid/hyperploid degeneration-prone neurons, interfering with neuronal function and plasticity.
Subsequently, it was also discovered that neuronal degeneration, primarily caused by Eg5 inhibition and subsequent mitotic spindle alteration, is further enhanced by inhibition of Eg5-mediated neurotrophin and neurotransmitter receptor transport to the cell surface (Figure 7).Indeed, as previously reported, Eg5 seems to participate in protein carriage through CARTS migration [11].In AD, the amount of two essential receptors for the correct development and function of the CNS, the Nerve Growth Factor/ Neurotrophin receptor (NGF/NTR) and the N-methyl-Daspartate receptor (NMDA), appears to decrease on the surface of neurons after Aβ accumulation; this efect is comparable to that observed after chemical inhibition of Eg5 by monastrol.At the same time, neurons appear less susceptible to NGF with a reduction in neurite outgrowth, as well as Ca 2+ entry into neurons regulated by the glutamatedependent NMDA receptor [100].In addition, Aβ accumulation is responsible for inhibition of long-term potentiation (LTP) NMDA-dependent, a key process for memory acquisition.When comparing the efect of Aβ and monastrol on LTP of the hippocampal slices, Ronald and co-workers found that monastrol can simulate Aβ efects on synaptic loss and LTP.In this way, they confrmed that Aβ causes acute and chronic synaptic dysfunction through inhibition of Eg5 [101], adding one more piece to the knowledge of the mechanism by which Aβ accumulation afects neuronal function in the disease.
Aβ accumulation is not the only cause of Eg5 inhibition and neuronal mitotic defects.In fact, as previously reported, the second main lesion recorded in AD patients is due to the formation of NTF by the hyperphosphorylated Tau protein.Tau is one of the main brain microtubuleassociated proteins (MAP), predominantly in axons and neurons, with the main function of stabilizing the structure of MTs.Tis protein is highly expressed and phosphorylated not only in AD patients, but also in other neurodegenerative diseases, defned as tauopathies.A French research group from INSERM U1191 found that in AD Tau excess alters normal Eg5 function, with alteration in the mitotic process (monopolar spindle formation) and aneuploidy/apoptotic neuronal cell death [102].Using a model of Drosophila developing wing disc epithelium (a columnar epithelium that during larval stages heads for several cell divisions to form future adult wing), they demonstrated that an excess of Tau protein induces mitotic arrest and monopolar spindle formation through a defect in the C-terminal Tau microtubule binding domain and an incorrect association of MTs during cell mitosis.However, they also found that these alterations in mitotic occurrence are due to specifc mitotic kinesin, the Klp61F, the Drosophila homologue of human Eg5.Excess neuronal Tau afects the correct Klp61F location on MTs, inducing mitotic alterations.Tis result has been confrmed in an in vitro human model represented by epithelial HeLa cells, where the efect of Eg5 alteration, under Tau excess conditions, was also demonstrated [102].
Based on this knowledge, a recent study focused attention on the possibility of increasing Eg5 expression in AD to overcome its loss Tau/Aβ-mediated (Figure 8).
In rat primary neurons cell culture, KIF11 overexpression, obtained by transient transfection, strongly decreased the Aβ-mediated spine loss.In an in vivo model, represented by AD mouse model overexpressing KIF11, spatial learning and LPT defcits have been averted, improving learning and memory abilities, thanks to Eg5 upregulation.However, these positive efects after Eg5 overexpression do not decrease Aβ deposition.Terefore, Eg5 is a new possible target for drug development in AD: in particular, prevention of its complete inhibition by Aβ accumulation strengthens brain function, although the presence of deposits in AD brain still represents the main critical point of the disease [103].

Genetic Conditions Involving KIF11 Mutations.
As previously specifed, Eg5 is encoded by the KIF11 gene, located on the 10q23.33chromosome.KIF11 alterations are responsible for some specifc conditions, such as microcephaly and retinopathies.In particular, genetic mutations within KIF11 gene are responsible for two specifc conditions: an autosomal dominant inherited disease characterized by microcephaly with or without chorioretinopathy, lymphedema, or intellectual disability (MCLID) and a familial exudative vitreous retinopathy (FEVR).
MCLID is a rare autosomal dominant condition described for the frst time in 1992 [104].In 2012, the association between this condition and KIF11 was reported for the frst time: heterozygous KIF11 mutations were found in 15 of the 20 families afected by the disease, in particular two nonsense, two splice sites, four missense, and six indels causing frameshift mutations, were identifed [105] and, subsequently, in 2014, fve novel heterozygous mutations were detected [106].
From a phenotypic point of view, patients with MCLID present a diferent expression of symptoms: in a cohort of 37 individuals from 22 families, 86% were afected by microcephaly, 78% had an ocular abnormality, 46% had lymphoedema of the lower extremities, 73% had mild to moderate learning difculties, 8% were afected by epilepsy and 8% had cardiac anomaly, demonstrating that there is a very high variability of phenotypic alterations [107].Te last update on this topic was reported in 2018 when, for the frst time, a microdeletion was identifed encompassing the entire KIF11 gene.It has been proven in two siblings, a boy and his sister, and in his father, all with true microcephaly, mild intellectual disability, and chorioretinopathy, at different levels [108].Biochemistry Research International Te function of generated Eg5 protein in genetic MCLID KIF dependent gene is, of course, compromised: the microcephaly symptom could be due to the reduced activity of Eg5 in spindle formation, being this protein essential for organogenesis in general [3] and for neural development in particular [109].However, the importance of KIF11 activity in the development and maintenance of retinal and lymphatic structures was highlighted by the fact that mutations in this gene were also detected in patients with chorioretinopathy and/or lymphedema, in addition to the microcephaly condition, supported by the fnding that KIF11 is also localized in the inner segment and ciliary compartments of murine retina photoreceptor cells [110].
FEVR is the second genetic condition highly related to KIF11 mutations.It is a hereditary disease characterized by anomalous retinal vascular development with avascular peripheral retina.Te consequence is a retinal vasculogenic disorder, with peripheral retinal nonperfusion, retinal folds, subretinal exudation, and detachment [111].In 2014, 4 heterozygous mutations in the KIF11 gene were identifed [109].Other seven genes could be mutated and involved in FEVR pathogenesis (LRP5, FZD4, TSPAN12, NDP, CTNNA1, CTNNB1, and ZNF408); for this reason, Wang et al. recruited a cohort of patients with KIF11 mutations related to FEVR (35 patients from 25 families) to compare the KIF11-dependent FEVR phenotype to the FEVR patient phenotype with mutations in other possible genes (39 patients).Important evidence emerged from this study: 80% of patients with FEVR-related KIF11 mutation (20/25) showed variants of function loss, while 48% (12/25) of the variants were de novo.Phenotypically, chorioretinal dysplasia emerged in 44.2% of patients with the FEVR-related KIF11 mutation, while only 1.3% with other FEVR-related gene mutations.An increase and straightening of peripheral vessels (ISPV) was observed in 17.1% when KIF11 mutated and in 50% of FEVR with mutations in other genes.From this analysis, it was possible to conclude that chorioretinal dysplasia is the dominant phenotype in KIF11-associated retinopathy, while ISPV is common in FEVR with mutations in other genes [111][112][113][114].
Undoubtedly, KIF11 plays a role in retinal vascular development and its mutations alter the normal process.Tis could be associated with three important evidences: (1) Eg5 is correlated with angiogenesis, in cancer feld, as previously described [13,47,66], but also in other noncancerous situations, such as retinopathy conditions; (2) Eg5 activity is essential for organogenesis.FEVR is one of the several pediatric vitreoretinopathies that can appear at birth   Severe exudative vitreoretinopathy as a common feature for CTNNB1, KIF11, and NDP variants plus sector degeneration for KIF11 Yang et al. [114] 2022

Biochemistry Research International
Update on the phenotypic and genotypic spectrum of KIF11-related retinopathy Wang et al. [113] 2022 Phenotype-based genetic analysis reveals missing heritability of KIF11-related retinopathy: Clinical and genetic fndings Chang et al. [135] 2023 Mutations in the TSPAN12 and KIF11 genes in severe retinopathy of prematurity Sun et al. [136] 2023 14 Biochemistry Research International or later; this means that the activity of this protein is essential for a correct retinal development; (3) KIF11 is also localized in the murine retina, as previously specifed [110].
Although it is more common to fnd information on Eg5 and cancer when searching in common research motors (PubMed, Scopus), there are many available papers in which Eg5/KIF11 mutations are associated with microcephaly, and in particular, with MCLID and retinal pathological conditions, such as FEVR.In Table 2, a list of publications of the last 10 years is provided mainly focused on the genetic alterations of Eg5 and its pathological implications.

Other Noncancerous
Diseases.Few articles associate other specifc pathological conditions with Eg5 role; for each of them, only few scientifc information is available, suggesting that further and deeper elucidations are required to ofcially associate Eg5 with other noncancerous diseases.One condition in which Eg5 appears to be involved is immune system alterations induced by human immunodefciency virus type 1 (HIV-1).HIV is known to be responsible for acquired immune defciency syndrome (AIDS) due to loss of CD 4+ T-lymphocytes, with the consequent defects in immune system activity.Tat transactivation factor is one of the main and important proteins produced by HIV to regulate its proliferation, but also to induce apoptosis in CD 4+ T-lymphocytes [137].Liu and colleagues in 2014, by using two in vitro models of Jurkat cells and primary CD 4+ T-lymphocytes from healthy donors, proposed a mechanistic explanation, Tat-Eg5 activity-mediated, by which HIV induces the disruption of T-lymphocytes [138].Tey found that Tat, through its lysine 85 at the carboxyl terminus, acts as an allosteric inhibitor of Eg5 in CD 4+ T-lymphocytes: by binding the allosteric site of Eg5, Tat inhibits protein ATPase activity through a block of ADP release and, therefore, determining a block of the entire ATP cycle, essential for Eg5 motor activity.As previously described, Eg5 activity inhibition provokes a block of the mitosis process, with formation of a monopolar spindle that culminates in apoptosis triggering.Tis happens also in CD 4+ T-lymphocytes when Tat blocks Eg5 activity: the formation of the bipolar spindle is impaired with a block in cell cycle progression and apoptosis induction.A second study identifed Eg5 as an essential driver of natural killer (NK) response to Cryptococcus infection in HIV patients.Cryptococcus is the main cause of fungal meningitis in HIV patients, and NK cells drive the cytolytic efect to remove it.Eg5, together with dynein, converge the NK cell granules, through MT structures, in the direction of Cryptococcus and controls the degranulation process, with the release of perforin, the main killing agent [139].
A novel bioinformatic analysis, aimed at identifying new possible biomarkers involved in pulmonary arterial hypertension, discovered KIF11 among the 10 hub genes involved in the disease, revealing that it is upregulated [140].Additionally, in a study published in 2021, a correlation was also found between Eg5 and COVID-19: KIF11 is among the key genes altered in blood cells from patients infected, compared to normal blood cells [141].

Conclusions
Tis review summarizes the literature on Eg5 expression and functions in diferent pathological conditions.Te main point emerging from this analysis is the dual efect of Eg5.In fact, this is the frst review in which the role of Eg5 is elucidated taking into account not only its role in cancer, but also in other noncancerous diseases, opening new therapeutic frontiers.
On one hand, Eg5 overexpression or hyperactivity is responsible for diferent cancerous conditions, such as lung cancer and gastrointestinal tract carcinomas, followed by tumors of the reproductive system and of the central nervous system.Tis highlights the strong link existing between cancer onset/progression and Eg5 overexpression, classifying this kinesin as a new possible biomarker and a new therapeutic target.On the other hand, loss of function of Eg5, due to genetic alterations or structural/functional impairment, is responsible for diferent diseases, such as AD, microcephaly, or retinopathies, as well as AIDS, in which the association with Eg5 is less elucidated, thus identifying new frontiers to exploit.

Figure 1 :Figure 2 :
Figure 1: Eg5 activity in mitosis.(a) Te correct activity of Eg5 (dark green) allows the overlapping of two polar microtubules at the equator of the cell and the separation of centrosomes at the opposite poles of the cell, thus obtaining a bipolar spindle pole formation.(b) Eg5 alterations or inhibitions determine monopolar spindle formation (monoaster).

Figure 3 :
Figure 3: Schematic representation of the aim of this review: summarizing recent fndings on the well-known activity of Eg5 in cancer and recent and old fndings on the less well-known role of Eg5 in other diseases.

Figure 5 :
Figure 5: (a) In normal conditions, miRNA-30a counteracts tumor onset and progression by inhibiting Eg5 synthesis.(b) In tumors, such as LUAD, the lnc-RNA VSP9D1-AS1 sponges miRNA-30a activity induces gene expression and protein synthesis of Eg5.Blue arrows in (a) and (b) represent Eg5 release within the cytoplasm.

Figure 6 :
Figure 6: TRFA4/Smarf2/Eg5 is a possible axis involved in the onset of breast cancer.TRF4A overexpression blocks Smarf2-mediated Eg5 ubiquitination, with a reduction in Eg5 degradation and its consequent accumulation in cells, promoting proliferation of breast cancer cells and inhibition of apoptosis.

Figure 7 :Figure 8 :
Figure 7: Efects of Aβ accumulation in AD patients and its inhibitory efect on Eg5 activity.

Table 1 :
Te table resumes articles from 2021 to 2023 underlining a correlation between Eg5 expression and tumor onset and progression.
[61]Evaluation of the TRIP13 level in breast cancer and insights into potential molecular pathways Lan et al.[50]2022 Kinesin Eg5 selective inhibition by newly synthesized molecules as an alternative approach to counteract breast cancer progression: an in vitro study Ricci et al.[51]2022Identifcation of hub genes associated with breast cancer using integrated gene expression data with protein-protein interaction network 2022 Te kinesin Eg5 inhibitor K858 exerts antiproliferative and proapoptotic efects and attenuates the invasive potential of head and neck squamous carcinoma cells Nicolai et al.[60]2022 Nonmetabolic functions of phosphofructokinase-1 orchestrate tumor cellular invasion and genome maintenance under bevacizumab therapy Lim et al.[61]2022An in silico approach to the identifcation of diagnostic and prognostic markers in low-grade gliomas Schematic summary of the main cancerous conditions in which high expression of Eg5 was found and reported in the literature between 2021 and 2023.

Table 2 :
List of publications of the last 10 years on the genetic correlation between KIF11 mutations and microcephaly/MCLI-D-retinopathies/FEVR.