Multifaceted role of primary cilia and ciliary proteins: A potential nexus for hedgehog signaling and prostate cancer.

1.
Introduction
The cell surface is a dynamic and intricate space that plays a pivotal role in maintaining cellular growth and homeostasis, serving as a mediator between the internal cellular machinery and the external environment [1][2]. Cell membranes are decorated with multiple organelles, such as cilia, and surface proteins like receptors, ion channels, and transporters, which enable cells to interact with their surroundings in a highly regulated fashion [2][3]. These components are essential for cellular communication, enabling the uptake of vital nutrients like amino acids, glucose, and lipids necessary for energy production, supporting metabolic processes, and maintaining cellular balance. Additionally, they help eliminate waste products and toxins, safeguarding the cell from damage [4][5][6]. These tightly regulated structures and their interactions at the cell surface ensure normal cellular function and also adaptability to stress or pathological conditions. Dysregulation of any of these surface structures/processes or cellular membrane homeostasis can lead to diseases, including cancer [7].
The primary cilium is an immotile, microtubule-based organelle that acts as a sensory hub, pick up signals from light, chemicals, or mechanical stimuli [8]. Molecular signals are received and transduced through the cilium, functioning as a specialized center for various signaling pathways, including Hedgehog (Hh), Notch, Wnt, receptor tyrosine kinases (RTKs), Hippo, transforming growth factor beta (TGF-β) receptors, and G protein-coupled receptors (GPCRs), which regulate cell behavior and tissue homeostasis [9][2][10]. Ciliary function and structure are governed through ciliary receptors, which are dynamically and precisely regulated by context-specific mechanisms, ensuring that cilia can respond accurately to environmental cues. Research has established that over a thousand proteins are localized at the primary cilium, and ciliary trafficking manages the movement and turnover of these critical proteins at the ciliary base, axoneme, and ciliary transition zone, which is essential for ciliary assembly and function [11][12]. Malformations and dysfunctions of primary cilia have been linked to a range of pathological conditions, including developmental disorders and cancer [13][14][15]. Recent progress in understanding cilia mechanisms has proposed that the primary cilium could act as a potential therapeutic target for drug discovery, attracting growing interest in cancer research, including PCa preinvasive and invasive stages [16].
Hedgehog signaling is a key developmental pathway that regulates cell proliferation, differentiation, and stemness. Canonical and non-canonical Hh signaling facilitate cell-cell communication and are crucial in determining cellular fate [17]. Generally, with a few exceptions, Hh-responsive cells are ciliated, and it is well established that the graded response to Hh signal transduction relies on the architecture of the cilia [18]. Both laboratory and clinical studies have highlighted the therapeutic potential of Hh signaling cascade by focusing on its regulatory components, particularly Smoothened (SMO) and Gli proteins. However, challenges remain, such as drug resistance and managing side effects, which researchers continue to address. Several studies have linked cilia and Hh signaling [19][20][21], but the precise roles of ciliary proteins in Hh signal transduction, particularly in the context of prostate cancer (PCa), remain understudied.
PCa is one of the frequently diagnosed cancers and the fifth leading cause of cancer-related death globally, predominantly impacting millions of men in high human development index regions [22]. PCa is a multifaceted and heterogeneous disease caused by the interplay of genetics, environment, and socioeconomic factors [23]. PCa patients generally respond well to the primary therapeutic options, whereas relapsed PCa are treated with androgen deprivation therapy (ADT) with a combination of chemotherapy or with second-generation AR-targeted therapies [24][25]. Managing disease relapse, resistance to castration or drugs, and metastasis presents major challenges to comprehending PCa pathology and treatment. Addressing these issues requires a comprehensive understanding of the altered molecular signatures and the modulation of signaling pathways. Recent evidence has demonstrated the potential of the ciliary-Hh pathway in various facets of PCa research. This review focuses on the critical role of cilia and their regulatory components in various cellular processes, including cell cycle regulation, cell death, and the oncogenic potential of cilia, with particular emphasis on their relationship with Hh signaling. Furthermore, we have highlighted how the molecular machinery involved in ciliogenesis influences the tumorigenic environment of the prostate and pinpoint gaps in our understanding of these interactions. We anticipate that addressing these existing scientific research gaps will help to illuminate both fundamental mechanisms and reveal potential therapeutic targets in PCa research.

2.
Primary Cilia and their influence on cellular function
2.1
Primary Cilia
The primary monocilium, or cilium, is an antenna-like structure that protrudes from the surface of most eukaryotic cells, playing a vital role in mechanosensation, cellular signaling, determination of cell polarity, cell homeostasis, and development [8][26][27]. The primary cilium consists of a basal body, axoneme, transition zone, ciliary membrane, diffusion barrier, and transition fibers (Fig. 1A). The basal body forms from a mother centriole, the axoneme is a microtubule-based structure covered by a distinct ciliary membrane, and the transition zone separates these two structures, often considered the “gatekeeper” of the cilium. The ciliary membrane surrounds the axoneme and is continuous with the plasma membrane. Together, the transition zone and transition fibers form a diffusion barrier that controls the movement of molecules between the cilium and the surrounding cytoplasm. Transition fibers connect the basal body to the plasma membrane, helping to anchor the cilium.
Cilia are broadly classified into two types: motile cilia and nonmotile cilia, which are also referred to as primary cilia. The primary cilia contain receptors and channels that detect external signals, such as mechanical flow and chemical stimulation, and transmit them into the cell [28]. The precise process of ciliogenesis and ciliary function enables cells to proliferate, migrate, and differentiate in a spatiotemporally controlled manner. In this context, ciliary alterations contribute to ciliopathies, which are characterized by diverse developmental abnormalities affecting multiple organ systems and have also been involved in the onset and advancement of cancers. [29][30]. Further evidence suggests that certain oncogenic signaling pathways and targeted anticancer therapies can either promote or suppress (loss and/or shortening) ciliation [31]. Collectively, the cilium is regarded as a “cellular watchtower,” and its absence potentially triggers the onset of neoplastic growth [32].

2.2
Ciliogenesis and Cell Cycle
Ciliogenesis is a multi-step process in which primary cilia typically form during the G1/G0 phase of the cell cycle and disassemble as cells re-enter the cell cycle (S phase) [33] (Fig 1B). The primary cilium formation in cells relies on one of the centrioles from the centrosome. Ciliary assembly proteins affect cell-cycle progression, and a centrosomal “mitotic kinase” facilitates ciliary disassembly [34]. Two distinct physiological pathways contribute to primary cilia assembly: an extracellular and an intracellular pathway [35]. In the extracellular pathway, the mother centriole first docks with the plasma membrane, leading to the nucleation of the ciliary microtubule doublet, named the axoneme. Whereas in the intracellular space, the axoneme begins to extend in the cytoplasm with an association of the mother centriole loaded with the ciliary vesicle [36]. Cilia disassembly is regulated in two diverse phases: the first phase generates signals for S phase entry, and once the cell is ready for the S phase, the second phase begins with shortening of the axoneme, releasing the basal body for mitotic spindle formation [37][36]. At the ciliary resorption stage, the disassembly of cilia is driven by the microtubule-depolymerizing actions of the Aurora A-HDAC6, Nek2-Kif24, and Plk1-Kif2A pathways [38][39][36]. Inturned and Fuzzy proteins, associated with the planar cell polarity pathway, regulate apical actin assembly, influencing the orientation but not the assembly of ciliary microtubules. Disruption of these proteins causes defective Hh signaling, resulting in impaired ciliogenesis [40].
Several lines of evidence suggested that the presence of primary cilia serves as a structural checkpoint for re-entry into the cell cycle [33][41]. Remarkably, ciliary length influences cell cycle time, and a study revealed that cell cycle-dependent mechanisms control ciliary length through a CDK5-SCFFbw7-Nde1 pathway [42]. The ubiquitin-proteasome system governs ciliogenesis in a cell cycle-dependent manner (Fig. 2A). In the early stage of axonemal extension, it removes trichoplein, a key negative regulator, from the mother centrioles, leading to Aurora-A inactivation and promoting ciliogenesis. Mechanistically, KCTD17 (K+ channel tetramerization domain-containing 17) has been identified through global E3 screening as a substrate-adaptor for Cul3-RING E3 ligases that polyubiquitylate trichoplein [43]. During the elongation phase, Nde1, a negative regulator of ciliary length, is ubiquitylated and degraded by CDK5-SCFFbw7 in a cell cycle-dependent manner [44]. Recent studies have highlighted that epigenetic modulations, including DNA methylation and histone/chromatin modifications, regulate cell cycle progression and cilia biogenesis [45]. These epigenetic mechanisms affect cell cycle regulators, including CDKs, cyclins, CDK inhibitors, and other factors such as Rb and p53 [46]. Since microtubules are a core component of the cilia axoneme, any factor that affects microtubule stability also influences ciliogenesis, such as epigenetic-regulator-mediated α-tubulin post-translational modifications [47]. Additionally, epigenetic changes in ciliary gene expression and cytoskeletal proteins further regulate ciliogenesis [48][45]. Furthermore, a study explored crosstalk between CDK4/6 and SMYD2 in microtubule dynamics, showing that their depletion or inhibition increased cilia assembly by altering microtubule stability and IFT20 expression, linking CDK4/6-SMYD2 signaling to ciliogenesis [49]. Moreover, non-canonical CDK6 activity was found to promote cilia disassembly by suppressing axoneme polyglutamylation [50]. Furthermore, a recent study showed that PI3Kα activation drives cilia disassembly via the PDK1/PKCι–CEP170–KIF2A axis, suggesting its role in ‘Disorders with Ciliary Contributions’, a subset of ciliopathies [51].

2.3
Ciliogenesis, Cilia and Cell Death
Cilia are integral in maintaining cellular homeostasis via regulating various cellular processes, including cell death. Cell death is a complex and multifaceted process, traditionally categorized into necrosis, apoptosis, and autophagy. Typically, necrosis was thought to be an uncontrolled, accidental form of cell death resulting from extreme damage or stress. However, advanced genetic research in the field of cell death has revealed that necrosis can be regulated in a programmed manner termed regulated necrosis/necroptosis [52][53]. Recent research has shown that loss of cilia from renal epithelial cells makes them more prone to necroptotic cell death during inflammation due to increased receptor-interacting protein kinase 1 expression, a key marker for necroptosis [54]. A recent study disclosed the significance of necroptosis in PCa, Shikonin, a natural compound, exhibited antitumor activity in both therapy-sensitive and docetaxel-resistant cell lines, primarily through necroptosis via enhanced pRIP1 and pRIP3 expression [55]. Furthermore, Curcumin, a polyphenol, concurrently triggers apoptosis and necroptosis in PCa cells by inducing mitochondrial oxidative dysfunction and ATP depletion. By investigating the role of cilia and ciliary-Hh signaling in necroptosis, a promising therapeutic target in PCa could be identified.
Apoptosis is a form of programmed cell death initiated through intrinsic (mitochondrial) or extrinsic (death receptor) pathways. Key players include caspase family proteases and Bcl-2 family proteins, which regulate mitochondrial permeability [56]. The connection between primary cilia and mitochondrion-mediated apoptosis underscores the significant role of cilia in cancer cell death (Fig. 2B). A study found that thyrocyte-specific loss of primary cilia in mice (Tg-Cre;Ift88flox/flox) and in human thyroid cancer cell lines, where KIF3A or IFT88 genes were silenced, led to increased apoptosis and Voltage-Dependent Anion Channel 1 (VDAC1) oligomerization, suggesting that loss of primary cilia serves as an apoptogenic stimulus [57]. VDACs regulate mitochondrial bioenergetics, including mitochondrial permeability transition and Bcl-2 family-driven cell death, and also negatively affect ciliogenesis by localizing to centrosomes [58][59]. A recent study showed that VDAC1 regulates tumorigenesis by modulating primary cilia disassembly and length, with its depletion increasing cilia numbers and length and reducing proliferation in pancreatic and glioblastoma cells [60]. Another study found that primary ciliogenesis influences the aggressiveness of atypical teratoid/rhabdoid tumors. Disruption of ciliogenesis reduces oncogenic potential, leading to decreased proliferation and increased apoptosis through activation of STAT1 and DR5 signaling [61]. However, the role of these ciliary-related molecular events and their association with Hh signaling in PCa remains an open area of research.
Beyond classical apoptosis, emerging research suggests that primary cilia may also regulate non-apoptotic cell death pathways such as ferroptosis and disulfidptosis. Studies suggested that NRF2 (Nuclear factor erythroid 2 2-related factor 2) plays a critical role in mitigating lipid peroxidation and ferroptosis [62][63]. One pivotal link between primary cilia and cell death is through the regulation of oxidative stress via NRF2, a master transcription factor that controls cellular antioxidant responses and redox homeostasis [64]. Furthermore, Hh signaling, which depends heavily on intact primary cilia, intersects with NRF2 pathways to modulate different cell death mechanisms. NRF2-dependent gene expression controls cilia formation and function and Hh signaling. NRF2-null cells exhibit fewer and shorter cilia and show disrupted Hh signaling, evidenced by the failure of Gli2 and Gli3 to localize at the ciliary tip upon Hh pathway activation [63]. Another study also supported that NRF2 negatively regulates primary ciliogenesis and Hh signaling through PTCH1. NRF2 hyperactivation promotes tumor progression via inhibiting Hh signaling and primary ciliogenesis via p62/sequestosome 1-dependent inclusion body formation and preventing Bardet-Biedl syndrome 4 entry into the cilia [65]. In PCa, oxidative stress is a known driver of tumor progression and therapeutic resistance, underscoring the relevance of the cilia-NRF2 axis. Disulfidptosis, a form of cell death characterized by actin cytoskeleton collapse under glucose starvation, which is dependent on the activation of the NRF2/SLC7A11 and NRF2/c-Myc axis [66][67]. Recent studies have indicated that disulfidptosis-related genes act as prognostic markers linked to tumor microenvironment (TME) features and immunotherapy response in PCa [67][68]. Given the central role of cilia in coordinating signaling pathways like Hh and NRF2, it is plausible that they modulate sensitivity to these alternative death mechanisms in PCa. The catabolic process, autophagy, is where cytosolic components such as damaged organelles, misfolded or aggregated proteins, and other cellular debris are encased in autophagosomes and transported to lysosomes [56]. A defined link between autophagy and ciliogenesis has been proposed, in which ciliary Hh signaling recruits autophagic machinery to initiate autophagosome formation, while autophagy, in turn, regulates ciliogenesis by controlling the destruction of ciliary proteins [69][70]. Several components of the autophagic machinery, such as ULK1, BECLIN1, VPS, and ATG machinery, localize at the axoneme and basal body of the cilium or in the periciliary region [71][70]. Additionally, ciliary proteins such as IFT88, IFT20, OFD1, PC2, ARL13, centrin1, and pericentrin are sequestered in autophagosomes [70]. Research into the interaction between primary cilia and autophagy has revealed a bimodal regulatory relationship (Fig. 2C, D). One study found that autophagy-mediated degradation of ciliopathy protein, oral-facial-digital syndrome 1 (OFD1) localized at centriolar satellites, promotes primary cilium biogenesis in both cycling and transformed cancer cells [72]. In particular, autophagy eliminated satellite OFD1, not centriolar OFD1, and suggested a positive role for autophagy in ciliogenesis. Conversely, another study reported that basal autophagy regulates ciliary growth by degrading proteins IFT-20, which are essential for intraflagellar transport, suggesting autophagy acts as a negative regulator for ciliogenesis [71]. Furthermore, a fine-tuned connection between the ciliary Hh pathway and autophagy has been noted, where Hh activation triggers autophagy by directly targeting key autophagy-related proteins at the cilium’s base. Activation of Hh signaling through Patched-1 knockout or Gli1 overexpression promoted autophagy by rescuing autophagy flux, whereas the inhibition via Smo knockdown or cyclopamine reduced starvation-induced autophagy [71]. This suggests that cilia upregulate autophagy through cilia-dependent Hh signaling. mTOR, a recognized negative regulator of autophagy, is activated in cilia-suppressed cells, leading to reduced autophagy. Inhibiting mTOR with rapamycin reversed autophagy suppression to shortened cilia, while upregulating autophagy activity prompted cilia elongation in kidney cells [73]. These findings acclaim the reciprocal regulation of cilia and autophagy through the mTOR signaling pathway and the ubiquitin-proteasome system. Remarkably, in retinal pigmented epithelium, glucose deprivation or mTORC1 inactivation increases the proportion of ciliated cells but shortens the cilium length by upregulating p27KIP1, not through autophagy [74]. In this manner, autophagy has a dual role in ciliogenesis, selectively turning cilia on or off by degrading ciliary essential or suppressive proteins. Interestingly, a recent study revealed that primary cilium-dependent lipophagy and mitochondrial biogenesis convert mechanical forces into metabolic adaptations in kidney epithelial cells [75]. However, it remains to be explored how ciliary Hh signaling components and ciliary elements influence or are influenced by autophagy regulation in PCa biology, presenting an emerging area of research.

3.
Significance of Cilia in Prostate Tumor Microenvironment Dynamics:
3.1
Role of Cilia in Prostate Cancer
The prostate originates from the prostatic anlagen of the urogenital sinus, with Hh signaling playing a critical role in guiding proper epithelial budding and ductal development [76]. During prostate development, Sonic Hedgehog (Shh) primarily functions through paracrine signaling, although limited autocrine activity can not be ruled out. In localized PCa, expression of Hh ligands and pathway activation are common and drive tumor cell proliferation through a combination of autocrine and paracrine mechanisms [77]. Notably, primary cilia were observed in both epithelial and mesenchymal cells during prostate development [78]. In PCa, the loss of cilia in cancer cells is linked to disrupted tissue homeostasis and increased malignancy [79]. A range of PCa cell lines derived from primary and metastatic sites lacked primary cilia [72]. Clinical and preclinical data reveal that cilia formation is often compromised in PCa, and restoring cilia is considered a potential therapeutic approach to mitigate PCa development (Fig. 3). The Spectral Karyotyping technique revealed that cancer/tumorigenic prostate epithelial cells often exhibit abnormal ploidy and reduced presence or length of primary cilia [80]. Interestingly, rapamycin, a potent inhibitor of mTORC1, restored primary cilia, increased ciliary length, and inhibited PCa cell proliferation [80]. The investigation of primary cilia function and frequency in preinvasive and invasive human PCa revealed that ciliated cell percentages in PIN, invasive cancer, and perineural invasion lesions decreased compared with normal tissue [81]. Additionally, the presence of shorter cilia across all stages of PCa was correlated with dysfunction and altered activity of the Wnt pathway. In this context, it is suggested that cilia normally suppress the Wnt signaling in epithelial cells, and their loss promotes Wnt signaling and supports PCa growth. Moreover, an association between cilia number and clinical parameters indicated that decreased cilium frequency was linked with larger tumor size [81]. The recent study also supported the idea that primary cilia influence PCa cell proliferation via the Wnt pathway. Rapamycin treatment restored the ciliated cells and reduced the β-catenin levels in PCa cell lines [82].

3.2
Cilia in Prostate Tumor Microenvironment:
As PCa advances, the reciprocal tumor-stroma interactions drive changes in the TME through intricate interactions between cancer cells and interconnected networks of stromal fibroblasts, immune cells, blood vessels, mesenchymal stem cells (MSCs), fat cells, and neural cells [83]. Single-cell gene expression and spatial transcriptomics have enhanced our understanding of the human prostate tumor microenvironment, revealing that it creates an immunosuppressive microenvironment characterized by enriched myeloid cells and Treg activity, exhausted CD56DIM NK cells and T-cells, activated B-cells, and high endothelial angiogenic activity [84]. A recent study also reported that a subset of localized PCa has an immunogenic phenotype. However, the prostate TME typically contains few tumor-infiltrating immune cells and low programmed death-ligand 1 (PD-L1) expression, classifying PCa as poorly immunogenic [85]. Cilia acts as cellular “watchtowers,” and their loss is proposed to be an early event in neoplastic transformation, functioning as a tumor suppressor organelle in some cancers, including PCa [79][86]. However, in some cancers, primary cilia are retained, serving as a hub for oncogenic signaling and therapy resistance [30][10][87]. In essence, the primary cilia’s impact on cancer is highly context-dependent. Oncogenic transformation can arise from disrupted ciliation dynamics, including cilia loss, structural defects, or altered length, all of which impact molecular signaling and cell cycle regulation [31][10]. Alterations in the ciliation of either cancer cells or other types of cells present in the TME promote asymmetric intercellular and paracellular signaling within the TME and regulate tumor growth and response to treatment [31][88]. The differences in signaling within all cells of the TME arise because many signaling rely on cilia for pathway activation. This disparity is attributed to heterogeneous ciliation in cancer cells and cells in TME: infiltrating lymphocytes and myeloid cells are typically non-ciliated, whereas fibroblasts and endothelial cells are more often ciliated compared to cancer cells [31][88].
Research on paracrine stromal Hh signaling in prostate tumorigenesis has revealed that primary cilia are present in human prostatic fibroblasts. These fibroblasts are capable of forming primary cilia and show active Hh signaling, as indicated by the co-localization of SMO at the tip of the primary cilium [89]. In the prostate tumor microenvironment, higher expression of Hh-related genes, such as Fgf5 and Igfbp6, in CAFs compared to adjacent non-malignant fibroblasts suggested that targeting the tumor stroma with anti-Hh therapies could be beneficial [89]. The study demonstrated a link between Hh signaling and stromal components, indicating variability of reactive stroma in advanced PCa. It also showed distinct stromal compositions across three PCa mouse models (PB-MYC, ERG/PTEN, and TRAMP), with elevated Hh signaling in smooth muscle cells and fibroblasts near tumors, while epithelial Shh decreased and Ihh and Dhh increased. Overall, Hh signaling was found to suppress tumor progression by preserving smooth muscle integrity and hampering micro-invasive PCa [90].
Paracrine Shh signaling promotes osteoblast differentiation, allowing Shh-expressing PCa cells to reshape the bone microenvironment and support metastatic growth [91][92]. Collagen produced by osteoblasts reinforces Shh signaling, creating a feedback loop that promotes osteoblast differentiation. In the presence of ascorbic acid, matrix collagen further enhances Shh-osteoblast interactions, and together they synergistically drive osteoblast differentiation, a central step in PCa bone metastasis [92]. A recent study highlighted the significance of osteocyte primary cilia in facilitating the migration of PCa cells [93]. Since bone metastases commonly cause suffering in PCa patients, this investigation revealed that osteocytes suppress cancer cell proliferation while enhancing migration through tumor necrosis factor-alpha (TNF-α) secretion. This mechanism is governed by osteocyte primary cilia and the associated IFT 88. Cancer cells hinder this mechanism by releasing TGF-β, which disrupts osteocyte cilia and IFT88 expression. These findings introduce a positive feedback loop in which PCa cells deactivate this anti-cancer mechanism, thereby promoting cancer cell proliferation, enhancing TGF-β production, and further suppressing osteocyte regulation.

4.
The Interplay between Cilia and Hh Signaling in Prostate Cancer
4.1
Cilia-mediated regulation of Hh Signaling
Hh signaling is transmitted through the canonical pathway, which relies on ligand-dependent interactions between receptors, as well as through non-canonical mechanisms that bypass the traditional Hh-SMO-Gli axis and operate independently of ligands, receptors, or Gli transcription factors [17]. This pathway activation is driven by three mature, lipid-modified ligands- Desert Hedgehog (Dhh), Shh, and Indian Hedgehog (Ihh), which are autocatalytically cleaved precursor proteins acting as soluble morphogens to direct specific cell fates [94][95][96]. These ligands bind to target cells via two main receptors: Patched1 (Ptch1), a 12-pass transmembrane protein, and SMO, a member of the F-class G protein-coupled receptor family [97][98]. In the absence of the signal, Ptch1 catalytically induces the constitutive suppression of Smo (Fig. 4A) [99]. SMO is mostly retained in intracellular compartments such as endosomes or the trans-Golgi network and adopts different inactive and active conformation that regulate its ciliary localization and movement [100]. β-arrestins, E3 ubiquitin ligases (e.g., Smurf), and other negative regulators promote SMO internalization, ubiquitination, and degradation or recycling. A recent study highlighted the ciliary protein Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and revealed the critical role of ciliary pocket-mediated endocytosis in cell signaling. Numb facilitates Ptch1 incorporation into clathrin-coated vesicles for its ciliary exit, a crucial step in Hh signaling. Loss of Numb blocks Shh-induced Ptch1 removal, leading to diminished Hh pathway activation[101].
Upon signal arrival at receiving cells, Hh ligands bind to Ptch1, triggering its exit from the primary cilium, and Hh–Ptch undergoes endocytosis. This relieves the inhibition on SMO, allowing it to accumulate in the primary cilium (Fig. 4A) [102]. SMO activation involves post-translational modifications causing conformational change and cytoplasmic tail dimerization [103][104] and possibly cholesterol/lipid binding, facilitating its ciliary trafficking through an intraflagellar transport (IFT) pathway [105][106]. Most events in reception of the Hh signal, such as biogenesis, release, reception, and intracellular transduction, occur at the primary cilium and require sterol modification (Fig. 4) [17]. The repression of Smo by Ptch1 occurs in cilia, and upon pathway activation, Smo accumulates in the ciliary membrane [20]. Activated SMO facilitates the nuclear translocation of Gli1, Gli2, and Gli3, leading to the transcription of target genes [107][108]. Gli proteins undergo proteolytic processing, where Gli1 acts mainly as an activator, while Gli2 and Gli3 function as both activators and repressors [109][110][111]. The trafficking of Hh pathway machinery through cilia relies on a set of motor and adaptor proteins [105]. Studies indicate that Sufu is also a key regulator protein required for expelling Gli from the nucleus and acts as a Gli repressor by interacting with Gli [19]. In the unstimulated state of Hh pathway, Sufu sequesters Gli transcription factors at the tip of the primary cilium [112]. The molecular basis through which Smo and Sufu regulate Hh pathway activation and repression, respectively, as well as the processes controlling the sub-ciliary localization of the Hh pathway components, remain critical areas of research, with many aspects still unresolved or unexplored [19] Within the primary cilium, both positive and negative effector molecules of the Hh pathway are processed via posttranslational modifications [113]. Canonical Hh signaling pathways, which rely on ligand-dependent activation, are entirely dependent on cilia. Conversely, non-canonical Hh pathways, activated independently of ligands, may exhibit either ciliary dependence or independence [113]. Non-canonical activation of Hh signaling in PCa cells has emerged as a key driver of castration resistance and advanced disease progression. This process is mediated by the interaction between transcriptionally active AR proteins and Gli3, with recent findings highlighting the oncogenic role of truncated Gli3 through the Gsk3β/Gli3/AR-V7 axis in castration-resistant prostate cancer [114][115]. With this evidence, vertebrate Hh signaling mechanisms are closely linked to primary cilium biology, highlighting how cilia-restricted signaling and ciliary dynamics regulate the spatial and temporal aspects of the Hh cascade [20].
Furthermore, mutations can also drive activation of the Hh pathway through cilia-dependent or independent mechanisms [113]. Firstly, mutations in Hh pathway proteins located upstream of cilia typically depend on cilia for regulation, such as the deactivation of Ptch1 and the constitutive activation of SMO. Secondly, mutation-driven activation of the Hh pathway can occur independently of cilia. This is due to mutations in Hh components located downstream of cilia, such as mutations or loss of Sufu or mutations or amplification of Gli, which enable the Hh pathway to be activated even in the absence of cilia [113][116]. Cilia act as distinctive signaling organelles and represent a dual role that can facilitate or impede Hh-dependent tumorigenesis, depending on the oncogenic initiating event [117]. At the molecular level, the cilium typically inhibits the Hh pathway by facilitating the formation of Gli3-R in the absence of Hh. However, the cilium is also necessary for SMO-mediated pathway activation in the presence of Hh. This brings up the question of how mutations alter the function or movement of cilia. Gaining insights into how these genetic changes impact ciliary dynamics could be crucial for understanding the mechanisms behind PCa progression.

4.2
Ciliary Hh Signaling: A Paradigm for Prostate Tumorigenesis
Paracrine Hh signaling responsiveness in the prostate correlates with primary cilia on stromal cells [78]. Research utilizing the urogenital sinus mesenchymal cell line UGSM-2 demonstrated that growth-restricting conditions, such as serum deprivation or cell confluence, induce the formation of cilia [78]. This phenomenon was associated with Hh pathway activation, linked to SMO accumulation in primary cilia. The relative contributions of autocrine and paracrine Hh signaling in PCa growth, metastasis, and drug resistance are crucial for PCa research. Earlier reports suggested the lack of demonstrable autocrine Hh signaling by the Shh ligand-dependent mechanism in human PCa cell lines PC-3, LNCaP, and 22Rv1 [118]. Surprisingly, while these cell lines exhibited activation of Hh target genes upon transfection with an activated version of Gli2, none showed a noticeable transcriptional response to either Hh ligand stimulation or transfection with an activated SMO. Notably, the same research group also investigated that these PCa cell lines, known to lack autocrine Hh signaling activity, did not manifest primary cilia even under growth conditions that restricted proliferation [78]. Hence, the lack of cilia in various PCa cell lines and their diminished response to Hh signaling suggest that cell-autonomous Hh signaling is probably not involved. This finding plausibly explains why Hh pathway activation can occur through the expression of activated Gli2 but not activated SMO [78]. A recent study highlighted that the secretory protein semaphorin 3C (SEMA3C) induces androgen synthesis in prostatic stromal cells through paracrine signaling. Accumulating evidence highlighted the potential of Hh signaling components in pre-clinical and clinical applications for PCa patients, though the direct link between cilia, the Hh pathway, and PCa remains an area of ongoing research.

5
Influence of Sterols on Cilia Function and Hh Signaling in Prostate Cancer
Sterols, including cholesterol, lipids, and fatty acids, are essential for ciliary function, Hh pathway integrity, and ciliary function [55]. High-fat diets and obesity are linked to increased PCa risk, and most human PCa cells exhibit a lipogenic phenotype, fueling tumor growth and aggression through disrupted lipid metabolism and signaling pathways [119][120][121][122]. Remarkably, PCa aggressiveness is associated with a distinctive metabolic signature marked by elevated de novo fatty acid synthesis, driven by activation of AR and coordinated by sterol regulatory-element binding proteins (SREBPs), c-Myc, PTEN/PI3K/Akt, and AMP-activated protein kinase [123][124][125][126]. Analysis of the PCa lipidome in clinical tissues has highlighted enhanced fatty acid synthesis, elongation, and desaturation as defining tumor characteristics. Targeting the mediators of these lipidomic alterations offers new therapeutic opportunities using current metabolic agents [127].
Research on tumor-associated lipogenesis revealed that activating the lipogenic pathway disrupts epithelial cyst formation, impairs ciliogenesis, and interferes with normal prostate gland development in mice. [128]. Phenotypically and mechanistically, an enhanced fatty acid supply leads to significant cell flattening and mislocalization of apical proteins [128]. Interestingly, inhibiting fatty acid synthesis by knocking down fatty acid synthase (FASN), a transcriptional target of SREBP1, restored primary cilium formation in highly lipogenic human PCa cells. This indicates that tumor-associated lipogenesis impairs cilium formation, affecting environmental sensing, signaling, and tissue architecture [128]. Moreover, ectopic FASN expression in the prostate of transgenic animals led to prostatic intraepithelial neoplasia, indicating that FASN acts as an oncogene in PCa, particularly in the presence of AR. Mechanistically, FASN promotes its oncogenic effects by blocking the intrinsic apoptosis pathway [129]. Further, another study reported that PTEN inactivation and PI3K/AKT pathway activation are key drivers of increased fatty acid synthase protein levels in LNCaP cells [126].
Adipose tissue and cancer cells coordinate a complex interaction network that fosters tumor growth and progression through extracellular vesicles EVs. These EVs carry enzymes and fatty acids that stimulate fatty acid oxidation, regulating mitochondrial metabolism and remodeling in tumor cells [130][131]. A recent study revealed that adipocyte-derived EVs influence PCa cell traits, leading to increased proliferation, migration, and invasion by facilitating phenotypic and metabolic remodeling [131]. Free-fatty acid receptors, such as FFAR1/GPR40, FFAR2/GPR43, FFAR3/GPR41, and FFAR4/GPR120, are ciliary GPCRs that are present in the primary cilia of preadipocytes. The ciliary GPCRs activate Gα proteins similar to their receptors located outside the cilium, but some GPCRs may also mediate cilia-specific signaling pathways that differ from the extraciliary pathway. The study found that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 facilitates adipogenesis [132]. Mechanistically, omega-3 fatty acids activate ciliary FFAR4 by rapidly increasing cAMP production within the cilia. This rise in ciliary cAMP triggers EPAC signaling, CTCF-dependent chromatin remodeling, and the transcriptional activation of PPARγ and CEBPα, thereby initiating adipogenesis. The differentiation of preadipocytes into adipocytes is driven by adipogenic signaling through IR/IGF1R receptor activation at the ciliary base, which leads to the accumulation of CAV1- or GM3-positive lipid rafts and ultimately stimulates the PI3K/AKT pathway [133][134]. Hh-activated smoothened through Fibro/adipogenic progenitors (FAPs) cilia regulate adipogenesis and orchestrate the regenerative response to skeletal muscle injury. Ciliary Hh signaling restricts FAP conversion into adipocytes through TIMP3, a secreted metalloproteinase inhibitor that inhibits MMP14, thereby blocking adipogenesis [135].

6.
Ciliary Associated Proteins and Hh Signaling in PCa: Research Gaps
6.1
Ciliary Trafficking and Ciliogenesis:
6.1.1
IFT family proteins:
Ciliated cells rely on a specialized transport mechanism, the intraflagellar transport (IFT) system, to facilitate the movement of molecules from the cell body to the cilium [136]. This multimolecular machinery consists of a wide array of IFT proteins organized into the anterograde IFT-B complex and the retrograde IFT-A complex [136]. Fascinatingly, research conducted in the past decade reveals that the functionality of the IFT system extends beyond the primary cilium and plays a role in essential extraciliary functions, such as cell cycle progression, mitotic spindle orientation, vascular trafficking, cytoskeletal dynamics, autophagy, lysosome genesis, and the cellular degradation pathway [137][138][139][140][141]. Moreover, ciliary proteins are found to co-localize with and interact with components of the proteasome at the base of the cilium, suggesting their involvement in cellular proteostasis, not only limiting the ciliary assembly/disassembly, ciliary length, and signaling. Loss of ciliopathy-related proteins disrupts proteasomal clearance, causing an accumulation of paracrine signaling mediators, including components of the Hh pathway. This accumulation manifests as elevated levels of SuFu, Gli2FL, and Gli3FL, accompanied by a simultaneous reduction in Gli3R, which are typically targeted for degradation by the proteasome [142]. The study also revealed that loss of retrograde IFT motors disrupts SMO localization in the cilium and affects Gli activator and repressor function, along with Gli3 proteolytic processing, indicating the critical role of primary cilia as specialized signal transduction organelles coupling SMO activity to Gli3 protein biochemical processing [143].
As IFT proteins are integral to the Hh signaling pathway, their function within cilia is categorized into two models. The first model suggests their involvement solely in cilia generation, where the cilia produce a signal essential for a specific step downstream of Ptch1, SMO, and Rab23 in the Hh signaling pathway. The second model delineates two distinct functions: cilia formation and intracellular transport [144]. Mechanistically, these IFT proteins are deeply integrated and regulate Hh signaling at a step downstream of Ptch1 and SMO and upstream of Gli direct targets [145][144]. The balance between positive and negative regulators of Hh signaling at the cilium tip regulates Shh pathway activation, and their transportation into and out of the cilium relies on IFT proteins and IFT motor proteins [146]. Investigating the movement of Hh components, the IFT protein family emerges as a critical facilitator, highlighting its pivotal role. It has been reported that SuFu and SMO localize to the cilium through an IFT122-independent mechanism. Perturbing IFT-122 interrupts Hh signaling; for instance, the null allele of Ift122sopb allows primary cilia formation but failure of retrograde intraflagellar transport features. Depletion of IFT122 leads to the accumulation of Gli2 and Gli3 at the cilium tip under the control of KIF7 [146]. A recent study showed Ift27 mutant cells display abnormalities at multiple stages of the Hh signal transduction pathway, such as the unusual accumulation of SMO in nonactivated cilia, localization of Gli2, Kif7, and SuFu at the ciliary tip, and enhanced levels of Gli3-FL. Interestingly, the impact of IFT27 deficiency does not affect the activation of gene expression triggered by truncated Gli2, indicating that nuclear entry and transcriptional stimulation are not reliant on IFT27. Furthermore, based on a rapamycin-induced in-cell dimerization system to sequester IFT-B proteins at the mitochondria, it was observed that disruption of IFT did not impact SMO delivery to cilia, and suggested that SMO entering the cilium might not require IFT-B transport machinery [145]. Indeed, employing highly sensitive single-molecule tracking spatial/temporal precision demonstrated activation-dependent changes in SMO dynamics within cilia. The study observed that SMO predominantly translocates to the cilium via membrane diffusion, and binding events are regulated by Ptch1 [147].
Considering the role of IFT family proteins in PCa, no significant expression of the IFT20 protein was detected in PCa and its surrounding tissues[148]. However, the role of IFT20 has been demonstrated in colorectal cancer cell invasion and guidance by regulatingGolgi-associated microtubule polarization, including reorienting the Golgi apparatus towards the direction of invasion in leader cells [149]. IFT88, a subunit of the IFT-B complex, is considered a tumor suppressor. The loss of IFT88 suppresses the constitutively active M2 allele of SMO-induced tumorigenesis by inhibiting the primary cilia-mediated Hh pathway [150]. Tumors initiated by SMO rely on the cilium to transduce positive signals through Gli activators and to suppress negative signals such as Gli3-R. Interestingly, in situ analyses of skin revealed that SMO M2-induced upregulation of direct targets of Hh signaling molecules Gli1 and Ptch1 was blocked by ciliary ablation, showing the importance of cilia in SMO-mediated induction of Hh pathway downstream targets. Furthermore, loss of Ift88 accelerated the formation of GLI2ΔN-induced BCC-like lesions, confirming that the cilium specifically modulates the Hh-related tumor phenotypes. In constitutively active GLI2ΔN-derived tumors, the absence of the cilium prevents Gli3-R formation, allowing unopposed activation of downstream signaling [150]. However, there is no report regarding Ift88 and PCa, and exploration is needed.

6.1.2
Dual-specificity tyrosine-regulated kinase
Dual-specificity tyrosine-regulated kinase (DYRK2) is a ciliogenesis-related protein that controls the mitotic transition, ciliary length, and cilia morphology [151]. The deletion of DYRK2 interfered with the expression of Aurora Kinase A (AURKA) and other cilia disassembly genes, resulting in impaired ciliogenesis [152]. This kinase functions as a priming kinase, facilitating subsequent substrate phosphorylation by GSK3β [153]. Reports suggested that DYRK2 is a positive regulator of Hh signaling and required for the dynamic trafficking of Hh components within cilia and phosphorylates Gli2 and Gli3 on serine residues at the ciliary base in response to activation of the Hh pathway [152][154][155]. DYRK2 acts as a scaffold for E3 ligase complex, regulating the p53 tumor suppressor in response to DNA damage, with its stability and pro-apoptotic functions regulated by ATM, which inhibits the MDM2 ubiquitin ligase [156][157]. A kinome-wide screen highlighted that DYRK2 phosphorylates Gli2, triggering its proteasome-dependent degradation [158]. DYRK2 is considered a tumor suppressor and is involved in cell-cycle-driven cancer development by regulating proteins like c-Jun, c-Myc, Rpt3, TERT, and katanin p60 [159][153][160][161][156]. DYRK2 expression was remarkably diminished or abolished in prostate tumor samples [153]. Nonetheless, further research is required to explore the mechanistic role of DYRK2 in PCa pathology and potential therapeutic applications.

6.2
Centriole-protein in ciliary assembly
6.2.1
Arf family proteins
The ADP-ribosylation factor (ARF) family of guanine-nucleotide-binding (G) proteins, including the ARF proteins and ARF-like (ARL) proteins, is regulated through a tight spatial control cycle of GTP binding and GTP hydrolysis mediated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) [162]. ARF family proteins are primarily recognized for regulating many cellular functions, including vesicular trafficking, cytoskeletal dynamics, and organelle structure, by recruiting several coat proteins, monitoring phospholipid metabolism, and modulating the actin structure at membrane surfaces [163]. Emerging evidence has suggested the novel roles of ARF and ARL proteins at the Golgi complex for driving lipid transport and during ciliogenesis [162].
Arl13b is one of ~30 ARF family proteins and is considered a ciliary protein required to maintain the ciliary structure and regulate the cell cycle, migration, and signaling. Intriguingly, the post-translational modification of Arl13b through palmitoylation controls its localization, stability, abundance, and function, which is ultimately required for cilia formation. During cilia resorption, cells promote the degradation of Arl13b by depalmitoylating it [164]. Determining the role of Arl13b within the cilium with respect to the Hh signaling, the study suggested that the dynamics of Shh signaling component localization to the cilium is disrupted in the absence of Arl13b. Remarkably, SMO was enriched in Arl13b-null cilia regardless of Shh pathway stimulation, indicating that Arl13b regulates the ciliary entry of SMO. Hence, abnormal Shh signaling in Arl13b mutant embryos results from defects in protein localization and distribution within the cilium [165]. The hnn mutation is a null allele of Arl13b, resulting in hnn mutant cilia length was short with specific structural defects in the ciliary axoneme. This defect impairs the ability of cells to translate varying levels of Shh ligand into differential regulation of Gli transcription factors that implement the Hh signal. Double mutant analysis (Arl13bhnn mutants) indicated that Gli activators (GliA) are constitutively active without impacting Gli3 repressor (GliR) activity. That suggested that normal trafficking of Shh components to cilia is lost, resulting in Ptch1 and SMO being located in cilia regardless of the presence of Shh and no ligand-dependent Gli accumulation on the ciliary tip. This study interpreted that the graded response to Shh, i.e., production of the Gli activity gradient, which is required for the spatial organization of ventral neural cell types, depends on the cilia architecture [166]. Another earlier study revealed that cells with the defective Arl13b hennin (hnn) variant divide slowly and become arrested at the G2/M phase. Mechanistically, these cells exhibit decreased p-ERK1/2 signaling and increased Sufu protein levels [167]. Apart from these studies, it has also been reported that Arl13b functions outside of the cilium and participates in cilia-independent Shh-mediated axon guidance [168]. Arl13b is also considered a mechanosensitive molecule mediating osteogenesis and is required for Hh signaling activation in osteoblasts [168].
A study reported that ARF1 can be considered a critical molecular target for PCa therapeutics and diagnosis. It was noticed that ARF1 expression is significantly elevated in PCa cells and human tissues, and increased ARF1 is correlated with PCa cell proliferation, anchorage-independent growth, and tumor growth [169]. Studies reported that ARF6 expression is elevated in cancerous cells and correlated with high cancer invasion and metastasis. It has also been reported that ARF6 expression was significantly increased in PCa samples compared to normal prostate tissue, and staining/protein expression was associated with increased staining intensity at higher Gleason score [170]. However, this study cannot exclude the possibility that the ARF6 antibody used in samples may also detect other ARF proteins due to the high homology, with over 70% sequence identity, among the most divergent isoforms of the ARF GTPase family. ARL11, also known as ADP-ribosylation factor-like tumor suppressor protein 1 (ARLTS1), is a tumor suppressor gene located at 13q14.3. ARLTS1 variants, especially Cys148Arg (T442C), are associated with increased susceptibility to different cancers, including PCa risk [171][172]. Remarkably, chromosomal region 13q14 risk variants associated with differential ARLTS1 expression and decreased ARLTS1 expression in PCa cases were detected. Additionally, research has linked ARLTS1 expression to immune processes, identifying the ARLTS1 Cys148Arg variant as a key contributor to sporadic and familial PCa risk and a potential biomarker for aggressive disease outcomes [173].
AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat, and PH domain 2) is a member of the Arf GAP (Arf GTPase activating protein) family considered a proto-oncogene involved in signaling regulation. AGAP2 expression levels are elevated in PCa, and the transcription factor SP1 binds to the AGAP2 promoter, playing a crucial role in its expression [174]. A lentiviral vector-mediated insertional mutagenesis screen identified 11 candidate genes, including ARFGAP3, that affect androgen-independent PCa progression and predict clinical outcomes [175]. Furthermore, ASAP1 is an ARFGAP associated with the metastatic potential of PCa and represents a potential therapeutic target and/or biomarker for metastatic disease[176]. However, the connection between ARF1, ARF6, ARL11, AGAP2, ARFGAP3, and ASAP1 in relation to ciliary-mediated Hh signaling, along with PCa progression, requires investigation, marking it as an intriguing area of PCa research.

6.2.2
SCL/TAL1 Interrupting Locus
STIL (SCL/TAL1 Interrupting Locus) is also known as SIL, a ciliary-related protein, and is considered a crucial centriole duplication factor in human cells [177]. STIL cooperates with SAS-6 and PLK4 to control centriole number and mitotic spindle integrity, both of which are required for genome stability and ciliogenesis [178]. The aberrant excessive STIL expression in differentiated tissues is associated with centrosomal amplification, leading to accelerated cell cycle progression [177]. Duplication of centrosomes is a fine-tuned mechanism, and an error in this process increases the risk of aberrant chromosomal segregation and aneuploidy, i.e., abnormal chromosome content. Chromosome segregation errors occurring in mitosis, often abbreviated as mitotic aberrations and aneuploidy, are widely recognized as a distinctive feature of tumor cells, fueling tumorigenesis due to defects in mitotic checkpoint signaling [179][180]. Pathological perturbations of STIL expression result in chromosomal instability, and a notable positive correlation between STIL mRNA expression and aneuploidy score in pan-cancer patients has been noted [181][182]. Conversely, STIL expression negatively correlated with several proteins involved in cilia axoneme formation, the intraflagellar transport (IFT) family, and Bardet-Biedl syndrome (BBS) proteins[182]. Cilia axoneme and IFT family proteins are essential for ciliogenesis and maintenance of ciliary signaling compartments [183].
Emerging evidence suggests this centriole protein functions as an oncogenic driver in a cilia-dependent manner in cancer [184][182]. Gene Ontology (GO) enrichment analysis has highlighted the involvement of STIL in regulating various pathways, including cell cycle, mitotic spindle organization, G2M checkpoint, and E2F target pathways, across 33 cancer types. This suggests that regardless of the tissue origin of cancer, the mode of STIL molecular action in cancer pathogenesis remains consistent [182]. Findings indicated that STIL-depleted cells exhibited increased expression of IFT88 and diminished levels of critical components involved in Hh signaling, including Gli and Shh [182]. Furthermore, an elevated STIL expression landscape has been linked to the advancement of cancer, including progression to more advanced pathological stages, increased malignancy, and unfavorable prognosis. It is associated with the immune cells infiltration, immune checkpoint protein expression, and the efficacy of immunotherapies [182][185][186]. Remarkably, in PCa, increased STIL promotes tumor growth by regulating MAPK/ERK, PI3K/Akt, and AMPK pathways and correlates with high-grade PCa tissue [187]. Studies indicated that silencing STIL expression stimulated the primary cilia formation and inhibited cell cycle protein expression (CCNB1 and CDK1) in PCa cell lines. The SHH pathway is linked with primary cilia and relies on STIL for pathway activation [188].

6.2.3
Transforming acidic coiled-coil protein-3
Transforming acidic coiled-coil protein-3 (TACC3) is a centrosomal protein that regulates centrosome and microtubule dynamics. As a substrate of the mitotic kinase Aurora A, both TACC3 and Aurora A are crucial for central spindle formation and potentially influence primary cilium generation. Recent studies have revealed that TACC3 is significantly upregulated in PCa and is associated with metastasis, tumor stage, PSA levels, Gleason score, and poor patient survival [189][190]. Knocking down TACC3 suppresses prostate tumorigenesis, reducing migration and invasiveness by inhibiting the Wnt/β-catenin signaling pathway [189]. Additionally, another study reported that TACC3 interacts with filamin A, disrupting its interaction with meckelin, which in turn inhibits primary cilium formation in PCa cells [190].

6.3
Centriole-protein in ciliary disassembly
6.3.1
Aurora A
The mitotic AURKA is vital for mitosis and also participates in several unforeseen non-mitotic functions[191][192]. The AURKA has proven to be a crucial regulator of primary cilium disassembly and depends on interactions with Ca(2+) and calmodulin[193]. Dysregulation of AURKA noncatalytic function is known to contribute to the development and progression of various diseases, including cancer. Studies have found that androgen regulate AURKA expression in castration-resistant prostate cancer, and suppressing Aurora A is considered a promising therapeutic strategy for androgen-independent PCa, as it suppresses tumor growth and increases chemosensitivity [194][195][196]. Notably, the suppression of primary cilia formation in PCa is associated with increased expression of AURKA[190]. Earlier findings shown that KIF14 regulates ciliogenesis through Aurora A, linking cell cycle machinery to efficient ciliogenesis [197]. Moreover, deregulated Aurora A activity as a downstream mediator of KIF14 deficiency leads to defects in primary cilium formation, cilium elongation, basal body biogenesis, and Hh signaling. The functional consequences of KIF14-depleted cells failed to induce Gli1 expression and exhibited impaired SMO translocation into the primary cilia in response to SMO receptor agonist (SAG) treatment. However, in KIF14-depleted cells treated with the AURA inhibitor significantly rescued the percentage of ciliated cells and primary cilium length, while SMO accumulation in primary cilia remained impaired. The study suggests that KIF14 has a dual role in primary cilia formation and function, regulating Hh signaling independently of AURA activity. Nevertheless, these in-depth mechanisms need further investigation in the context of PCa.

6.3.2
Polo-like kinase 1
Polo-like kinase 1 (PLK1), a crucial cell cycle regulator, has been implicated in PCa progression and metastasis and linked to higher tumor grade [198]. Research has demonstrated that the overexpression of PLK1 in prostate epithelial cells induces oncogenic transformation, causing significant transcriptional reprogramming and driving the cells toward EMT transition via regulating ERK/MAPK pathway [199]. In addition, the study identified the role of PLK 1 in facilitating the loss of Pten tumor suppressor-induced PCa formation. Mechanistically, elevated Plk1 is required for Pten-depleted cells to cope with mitotic stress and survival, while reintroducing wild-type Pten into Pten-null PCa cells reduces their reliance on PLK1 for survival [200]. Interestingly, a recent study highlighted that elevating PLK1 lowers SPOP mutation-stabilized Bromodomain-containing protein 4 (BRD4) and overcomes bromodomain and extra-terminal inhibitor resistance via triggering BRD4 phosphorylation-dependent degradation in mitosis, ultimately re-sensitizing PCa cells to BRD4 inhibitors [201]. Next, in the context of the Hh pathway, it has been revealed that the Plk1 kinase negatively regulates the Hh signaling pathway by phosphorylating Gli1 at S48, and this modification facilitates the nuclear export of Gli1 and its interaction with the negative regulator Sufu [202]. Hence, PLK1 has been considered a potential therapeutic target for PCa.

6.3.3
NIMA-related kinase 2
NIMA-related kinase 2 (NEK2) is a serine/threonine-protein kinase that localizes to centrosomes and kinetochores, regulating centrosome duplication and spindle assembly checkpoints during mitosis [203]. In PCa, higher levels of NEK2 overexpression are positively correlated with Gleason score, pathological stage, and poorer prognosis [204]. Nek2A is recognized as a SuFu-interacting protein that stabilizes SuFu by preventing its ubiquitin/proteasome-mediated degradation [205]. A study also deciphered that Nek2A promotes the phosphorylation of SuFu, inhibiting the nuclear localization and transcriptional activity of Gli2/Hh signaling [206]. Thus, Nek2A acts as a crucial modulator of the Hh pathway by driving a negative feedback loop [205].

7.
Preclinical and clinical evaluation of Hh Signaling and ciliogenesis targeting therapies in PCa
Preclinical research significantly showed their potential to reduce tumor invasiveness and metastatic progression of PCa cells by interference with Hh-SMO-Gli signaling [207][208][209][115]. Consistent findings from genetically engineered mouse models (GEMMs), xenografts, and patient-derived xenograft (PDX) models indicate that aberrant activation of Hh signaling, particularly through SMO, drives prostate tumor progression, metastasis, and therapeutic resistance. In the pCX-shh-IG mice model, constitutive overexpression of Shh in the prostate epithelium leads to the rapid development of prostatic intraepithelial neoplasia (PIN), which progresses to invasive and metastatic prostate carcinoma within 90 days of age. Notably, these tumors exhibit features consistent with a cancer stem cell–like phenotype and display metastatic potential independent of AR signaling [210]. Additionally, the Smo inhibitor NVP-LDE-225 (Erismodegib) has been shown to suppress EMT and inhibit the growth of human PCa stem cells in NOD/SCID IL2Rγ-null mice. These effects were associated with reduced expression of Gli1, Gli2, Patched-1, Patched-2, and regulation of key apoptotic proteins. Darinaparsin, an organic arsenic compound, inhibits Hh signaling by suppressing GLI2 transcriptional activity and reducing prostate tumor-initiating cells and growth in DU145 xenograft models [211]. SMO antagonist, TAK-441, has been shown to delay castration-resistant progression in LNCaP xenograft models by interfering with paracrine Hh signaling within the tumor microenvironment[212]. Next, another SMO antagonist GDC-0449, attenuated Hh signaling as evidenced by reduced expression of Gli1 and Ptch1 in the bone-forming PCa xenograft [213]. In a recent study, vismodegib, an SMO inhibitor, effectively reduced EMT and tumor growth in CRPC models (C4–2B xenografts) following oral dosing (~50 mg/kg) [214].
Numerous synthetic and naturally occurring small-molecule Hh pathway inhibitors, including cyclopamine, SANT1, Cur-61414, Robotnikinin, GANT58, GANT61, Vismodegib (GDC-0449), Sonidegib (Erismodegib, NVP-LDE-225, LDE-225), Glasdegib (PF-04449913), Saridegib (patidegib, IPI-926), Taladegib (LY2940680), Itraconazole, BMS-833923 (XL139), LEQ-506 (NVP-LEQ506), and TAK-441 have been developed [215][216][217]. While some of them have entered clinical trials, but shown limited efficacy, though some are still being evaluated. Ongoing trials in PCa continue to investigate the clinical efficacy of Hh inhibitors, despite reports of acquired resistance in other cancers with prolonged use[21][217]. Most approved Hh inhibitors (e.g., vismodegib, sonidegib) target SMO, whose function relies on primary cilia, thus blocking cilia-dependent signaling. Resistance to SMO inhibitors, often due to mutations or structural and functional alterations of primary cilia, demands agents that act downstream, such as GLI inhibitors is an active area of clinical research. The table summarizes key Hh pathway inhibitors, their relationship to primary cilia, and representative clinical trials with respect to PCa. The inclusion of both cilia-dependent and cilia-independent inhibitors underscores the therapeutic diversity and the importance of tailoring interventions based on prostate tumor biology and potential resistance mechanisms.
As primary cilia, essential organelles for canonical Hh transduction, appear progressively lost in PCa [82]. In cancer biology, primary cilia have a dual and context-dependent tumor suppressive or tumor-promoting role [10]. Remarkably, primary cilia have been considered as a novel research approach to overcome anticancer drug resistance[87][218]. Although the targeting of ciliogenesis in PCa is still in its infancy, murine studies manipulating IFT proteins (IFT88) or regulators of ciliary disassembly (e.g., Aurora A kinase, NEK2) affect prostate tumor biology and signaling output via activation of AR [93][194][204]. KIF3a, a subunit of kinesin-II motor protein, expression levels of KIF3a correlate with a higher Gleason score, tumor-node-metastasis stage, and metastatic status of PCa. To date, no clinical trials have directly targeted cilia biogenesis in PCa patients; however, several key preclinical studies underscore its potential therapeutic relevance. Exogenous expression of KIF3a promoted cell growth in the benign prostate cells, whereas silencing KIF3a in cancer cells decreased cell proliferation, anchorage-independent cell growth, and cell migration/invasion in advanced PCa through the KIF3a-DVL2-β-catenin axis [219]. A most recent mechanistic study revealed that the hsa_circ_0005185/OTUB1/RAB8A axis plays a critical role in regulating primary ciliogenesis through the Hh cascade. At the molecular level, circular RNA hsa_circ_0005185 promotes OTUB1-mediated deubiquitination of RAB8A and stabilizes RAB8A, which restores ciliary formation, leading to production of Gli3R, which is an inhibitory signal for the Hh pathway and subsequently suppresses AR signaling, providing a potential route to impede CRPC progression[220]. The availability of potential chemical modulators/ drugs targeting cilia-associated proteins or restoring primary cilium expression in PCa remains very limited. Using a high content analysis-based approach, a diverse set of 118 compounds stimulating cilium expression was identified, including glucocorticoids, fibrates, and other nuclear receptor modulators, neurotransmitter regulators, ion channel modulators, tyrosine kinase inhibitors, DNA gyrase/topoisomerase inhibitors, antibacterial compounds, protein inhibitors, microtubule modulators, and COX inhibitors. and certain of them impacted on ciliary length [64]. Other modulators like Clofibrate, Gefitinib, Sirolimus, Imexon, and Dexamethasone exhibit ciliogenesis-restoring activity confirmed in a panel of human cancer cell line models of different cancer types (pancreas, lung, kidney, breast), but have not yet been tested or confirmed in PCa. Gefitinib and Jasplakinolide are the only compounds documented to actively induce ciliogenesis with Gli1/IFT20 signature in PCa model systems through YAP1 modulation, making them especially relevant for further mechanistic or translational studies [221].
In summary, in vivo models have provided strong evidence for the oncogenic role of Hedgehog signaling in PCa, and emerging data highlight the potential of ciliogenesis as a regulatory axis. However, while Hh pathway inhibitors have progressed to clinical trials, therapeutic targeting of primary cilia remains largely unexplored in patients. Future studies should aim to combine genetic and pharmacological tools in orthotopic and PDX models to validate the therapeutic relevance of ciliogenesis regulation in PCa progression and treatment resistance.

8.
Future Prospects:
Primary cilia serve as essential organelles that act as scaffolds and create specialized microenvironments necessary for transmitting Hh signals within cells [222]. For effective signal reception and transmission, proper cilium biogenesis and ciliary length are essential. Therefore, disruption of the regulatory trafficking network within the Hh pathway and of the molecular machinery involved in ciliogenesis is associated with tumorigenesis and can promote the aggressive traits of PCa. Understanding the contributions of autocrine and paracrine Hh signaling, which are affected by the ciliary molecular machinery, is critical for advancing PCa research. Direct links between cilia and immune modulation in PCa remain unexplored, offering an exciting research opportunity. Hence, exploring how restoring ciliogenesis or modulating ciliary Hh signaling could impact immune infiltration or treatment response may open new therapeutic avenues. A deep understanding of these mechanisms will provide a clearer picture of their roles in PCa, aid in the development of novel therapies, and facilitate personalized treatment strategies tailored to individual tumor profiles. Hence, the in-depth molecular mechanisms of ciliary Hh signaling and its crosstalk with ciliogenesis-related proteins in PCa remain a promising and open area for future research.