AhR-Siglec-15 axis regulates lysosomal Ca²⁺ release for sonic hedgehog medulloblastoma growth via TRPML1

Abstract

Sonic hedgehog subgroup medulloblastoma (SHH-MB), an aggressive pediatric brain tumor that originates from granule neuron precursors, faces the challenge of poor treatment owing to its unclear molecular mechanisms. Here, we show that sialic acid-binding immunoglobulin-like receptor 15 (Siglec-15), an immunosuppressive membrane protein, is upregulated and mediates SHH-MB growth through its translocation to the lysosomal membrane. We found that SHH-MB cells use the cation-independent mannose 6-phosphate receptor (CI-MPR) to transport Siglec-15 from the trans-Golgi network (TGN) to lysosomes, where Siglec-15 induces lysosomal Ca²⁺ release by interacting with mucolipin TRP cation channel 1 (TRPML1), leading to the nuclear translocation of the transcription factor EB (TFEB). Blockade of Siglec-15, TRPML1, or TFEB hinders SHH-MB growth in vitro and in vivo. Importantly, aryl hydrocarbon receptor (AhR), a cytoplasmic transcription factor, upregulates Siglec-15 expression. AhR inhibition by CH-223191 or StemRegenin 1 (SR1) achieved therapeutic efficacy against orthotopic SHH-MB xenografts in mice. These findings reveal an essential role for the AhR-Siglec-15 axis in SHH-MB development, providing a potential strategy for SHH-MB treatment.

Graphical abstract

Keywords

Siglec-15 / AhR / TFEB / lysosome / TRPML1

Introduction

Medulloblastoma (MB), a neuronally invasive embryonal malignancy, is among the most common cerebellar tumors in children (Northcott et al., 2019), and commonly arises in the vermis and projects into the fourth ventricle (Louis et al., 2016; Millard and De Braganca, 2016). Based on molecular characteristics, MBs have been classified into four subgroups: wingless (WNT), SHH, Group 3, and Group 4 (Louis et al., 2016, 2021; Orr, 2020). In WNT–MB patients, tumorigenesis is driven by activating β-catenin mutations that constitutively stimulate WNT signaling. In contrast, the most common genetic events in Group 3 and Group 4 are high-level amplifications of the MYC oncogene and overexpression of PRDM6, respectively (Northcott et al., 2017). The SHH subgroup, a major subtype that involves sonic hedgehog (SHH) signaling, which is essential for the development of the cerebellum and tumorigenesis, has been extensively investigated (Chen et al., 2022; Garcia-Lopez et al., 2021; Zhang et al., 2022). Patched 1 (PTCH1) is an inhibitor of SHH signaling. During cerebellar development, Purkinje cells secrete SHH, a major mitogen of cerebellar granule cell progenitors, into the external granule cell layer. By binding to the SHH ligand, PTCH1 relieves its inhibition of smoothened (SMO). Activation of SMO triggers downstream signaling events by releasing glioma-associated oncogene (GLI) transcription factors from suppressor of fused (SUFU), a negative regulator of the pathway. This allows GLIs to translocate to the nucleus and induce activation of SHH pathway target genes. The PTCH1 mutation derepresses SMO and activates GLI for MB formation (Wang et al., 2022; Zhang et al., 2022). Thus, targeting SHH signaling has been highlighted as a critical strategy for SHH-MB treatment. Notwithstanding this, clinical benefits remain limited, suggesting that alternative signaling pathways may also play an important role in SHH-MB tumorigenesis and are urgently required for identification.

Human sialic acid-binding immunoglobulin-like receptor 15 (Siglec-15) is unique among Siglec family members, because it has a minimal intracellular domain but carries a lysine residue in the transmembrane domain that links the adaptor protein DNAX activation protein 12 (DAP12) or DAP10, implying its functioning as an activating signaling molecule (Kang et al., 2020; Macauley et al., 2014; Smith and Bertozzi, 2021). Surprisingly, Siglec-15 has recently been shown to transduce immunosuppressive signals and to function as an immune checkpoint during cancer immunotherapy (Fan et al., 2019; Kang et al., 2020). In addition, Siglec-15 is widely expressed in human cancers and has a tumor-promoting effect (Cao et al., 2021a; Fan et al., 2019, 2021; Liu et al., 2021). Notably, Siglec-15 has been reported to localize to the cytoplasm with unknown functions (Angata et al., 2007). In this study, we found that Siglec-15, transactivated by the aryl hydrocarbon receptor (AhR), is a critical factor for SHH-MB cell growth. Siglec-15 was translocated from the Golgi apparatus to lysosomes via cation-independent mannose 6-phosphate receptor (CI-MPR), where Siglec-15 interacts with mucolipin TRP cation channel 1 (TRPML1) to mediate lysosomal Ca²⁺ release, thereby activating the transcription factor EB (TFEB) and promoting SHH-MB cell growth. These findings reveal an unknown molecular mechanism, by which SHH-MB cell growth is regulated, and identify AhR and Siglec-15 as molecular targets for SHH-MB treatment.

Results

Siglec-15 expression is required for SHH-MB growth

Previous studies have integrated 23 transcription datasets into a large MB and normal cerebellar gene expression database (Weishaupt et al., 2019). By analyzing this, we found that the expression of Siglec-15 was lower in the G3 and G4 groups, but was higher in the SHH-MB group than in the healthy control group (Fig. 1A). Using the Group 3 MB cell lines D283 and D341, we found that Siglec-15 was poorly expressed in D283 and D341 cells and Siglec-15 overexpression did not increase their proliferation (Fig. S1A–C). Then, we further used published data (Cavalli et al., 2017) to analyze SHH-MB patient survival and found that Siglec-15 expression was correlated with poor survival (Fig. 1B), suggesting that Siglec-15 may have a SHH-MB-promoting effect. By analyzing the single-cell sequence data of MB patients (Riemondy et al., 2022), we found that Siglec-15 expression in SHH-MB tumor cells was the most abundant, compared to other subgroup MB cells and immune cells (Fig. S1D and S1E). In line with this, Siglec-15 was decently expressed in the Daoy SHH-MB cell line and primary SHH-MB tumor cells isolated from patients with SHH-MB, compared to the U87 glioblastoma cell line, which is known to highly express Siglec-15 (Fan et al., 2019) (Figs. 1C and S1F). By transfecting Siglec-15 shRNA into Daoy and primary SHH-MB tumor cells (Figs. S1G and S1H), we found that cell growth was reduced and cell viability was impaired (Fig. 1D and 1E). In contrast, Siglec-15 overexpression increased cell proliferation (Figs. 1F and S1I). ONS-76 is another SHH-MB cell line that expressed low levels of Siglec-15 (Fig. 1C). As expected, Siglec-15 overexpression promoted ONS-76 cell growth (Fig. S1J and S1K). By determining cell apoptosis and cell cycle in vector- and Siglec-15-overexpressing Daoy cells, we found that Siglec-15 drove cell cycle progression toward mitosis, but had no effect on apoptosis (Fig. S1L and S1M). To validate these results in vivo, we constructed Siglec-15 knockdown and firefly luciferase-expressing Daoy cells, and injected them into the cerebella of BALB/c nude mice (Fig. 1G). We found that Siglec-15 knockdown markedly inhibited tumor growth, reduced weight loss, and prolonged mouse survival (Fig. 1H–L). As expected, cerebellar inoculation of Siglec-15-overexpressing Daoy cells accelerated orthotopic tumor growth and shortened the long-term survival of mice (Figs. 1M and S1N–S1P). In addition, immunostaining showed Siglec-15 expression was higher in SHH-MB patient samples than in normal controls (Fig. 1N). Together, these results suggest that Siglec-15 has an SHH-MB-promoting effect.

Siglec-15 localizes to the lysosomes to mediate Ca²⁺ release

Next, we investigated the molecular basis of Siglec-15-promoted SHH-MB growth. In addition to the plasma membranes, Siglec-15 has been reported to be present in the cytoplasm (Angata et al., 2007). Using Siglec-15-overexpressing Daoy cells or primary SHH-MB tumor cells for immunostaining, we found that approximately 60% of Siglec-15 localized in the cytoplasm, while the remaining 40% was found on the plasma membrane (Fig. S2A). Ultra-high super-resolution microscopy showed that Siglec-15 did not colocalize with the mitochondria or endoplasmic reticulum (ER) and was slightly colocalized with the endosomes and Golgi apparatus, but mainly with the lysosomes in Daoy and ONS-76 cells (Figs. 2A and S2B–D). Consistently, western blot analysis showed that the majority of Siglec-15 protein molecules were present in the lysosomes (Fig. 2B). This unexpected lysosomal localization prompted us to speculate that Siglec-15 mobilizes lysosomes for tumor promotion. The mTOR signaling pathway is lysosome-dependent (Saxton and Sabatini, 2017) and exerts a tumor-promoting effect. However, transfection with Siglec-15-flag or Siglec-15 knockdown did not alter mTOR activity in Daoy and ONS-76 cells, excluding that Siglec-15 uses mTOR for tumor growth (Figs. 2C, S2E and S2F). Lysosomes are characterized by their acidic pH, which is also involved in tumor progression (Steffan et al., 2010). Again, Siglec-15 did not alter lysosomal pH in Daoy and ONS-76 cells (Figs. 2D and S2G). We then examined lysosomal Ca²⁺ release into the cytosol, which might augment cell proliferation (Cao et al., 2021b). Intriguingly, cytosolic Ca²⁺ levels were elevated in Siglec-15-flag-tranfected Daoy and primary SHH-MB tumor cells, compared to those in control cells (Figs. 2E, 2F, S2H and S2I). Knockdown of Siglec-15 reduced cytosolic Ca²⁺ levels, suggesting that Siglec-15 promotes the release of Ca²⁺ from organelles into the cytosol (Fig. 2G and 2H). In line with this notion, use of ryanodine, which blocks ER Ca²⁺ release, did not affect Siglec-15-increased Ca²⁺ levels in the cytosol (Figs. 2I, 2J, S2J and S2K). Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca²⁺ signals (Moccia et al., 2021). By treating the cells with GPN, we found that the calcium levels in the cells also increased (Fig. S2L and S2M). These results suggest that Siglec-15 localizes to the lysosomes to mediate Ca²⁺ release. TFEB, a master regulator of lysosomal biogenesis, autophagy, and signaling pathways, may be upregulated in tumor cells and is associated with tumor growth and proliferation (Franco-Juárez et al., 2022; Settembre et al., 2011). Notably, lysosome-released Ca²⁺ can activate TFEB effectively (Chen et al., 2018; Wei et al., 2023). In line with this, knockdown of Siglec-15 dampened the translocation of TFEB into the nucleus (Fig. 2K), concomitant with increased TFEB phosphorylation in Daoy and primary SHH-MB tumor cells (Figs. 2L and S2N); however, this effect was rescued by Siglec-15 overexpression (Fig. 2M). By knocking down TFEB (Fig. S2O and S2P), we found that Siglec-15-promoted Daoy cell viability was reduced (Fig. 2N). By injecting TFEB-knockdown and Siglec-15-overexpressing Daoy cells into mice, we found that TFEB knockdown counteracted the promoting effect of Siglec-15 on tumor growth (Fig. 2O and 2P). Together, these results suggest that Siglec-15 localizes to the lysosomes to mediate Ca²⁺ release and subsequent TFEB activation, thus promoting SHH-MB cell growth.

Siglec-15 interacts with TRPML1 to release lysosomal Ca²⁺

Next, we explored the manner by which Siglec-15 promoted lysosomal Ca²⁺ release. Lysosomal Ca²⁺ release can be mediated by two-pore channels (TPCs), purinergic receptor X4 (P2X4), and mucolipin subfamily of transient receptor potential channels (TRPMLs) (Cao et al., 2021b; Li et al., 2019). We found that knockdown of TPC1, TPC2, or P2X4 did not alter the effect of Siglec-15 on lysosomal Ca²⁺ release (Fig. S3A–D); however, the knockdown of TRPML1 or inhibiting TRPML1 by ML-SI3 blocked Siglec-15-promoted Ca²⁺ release, suggesting that Siglec-15 may target TRPML1 for lysosomal Ca²⁺ release (Figs. 3A, 3B and S3E–G). Co-immunoprecipitation (Co-IP) showed that TRPML1 was pulled down (Fig. 3C), suggesting an interaction between Siglec-15 and TRPML1. A consistent result was obtained from ultra-high super-resolution microscopy (Fig. 3D). To confirm this interaction, we deleted various domains and constructed different Siglec-15 mutants, in which the original signal peptide was included (Fig. 3E). We found that the deletion of the cytoplasmic domain or transmembrane/cytoplasmic region did not alter the interaction of Siglec-15 with TRPML1 (Fig. 3F and 3G), suggesting the N-terminal extracellular domain (ED) mediates the binding. Given that TRPML1 is a six-transmembrane protein with the N and C termini facing the cytosol (Fig. 3H), we constructed the intraluminal four loops (termed as E1–E4) of TRPML1, respectively. By performing bio-layer interferometry (BLI), we found that the recombinant Siglec-15-N (263 AAs) directly interacted with E1 but not E2, E3, or E4 (Figs. 3I, S3H and S3I). By performing glutathione S-transferase (GST) pull-down assay, we additionally verified that TRPML1 used its E1 domain to interact with Siglec-15 (Figs. 3J, S3J and S3K). In accordance with this, the knockdown of TRPML1 increased TFEB phosphorylation in Daoy cells (Fig. 3K). By injecting TRPML1-knockdown and Siglec-15-overexpressing Daoy cells into mice, we found that TRPML1 knockdown counteracted the promoting effect of Siglec-15 on tumor growth (Fig. 3L and 3M). Together, these results suggest that Siglec-15 binds to TRPML1, thus opening the calcium ion channel for lysosomal Ca²⁺ release.

CI-MPR mediates Siglec-15 transport from the Golgi to the lysosomes

Siglec-15 is a well-known transmembrane protein that prompted us to explore the pathway through which Siglec-15 is translocated to lysosomes, where it interacts with TRPML1. Endocytosis is a general process that involves translocation of plasma membrane proteins into lysosomes. As shown in Fig. S2B, Siglec-15 colocalized with the endosomes and Golgi apparatus. However, the use of various endocytosis inhibitors did not alter Siglec-15 localization to the lysosomes (Fig. S4A), suggesting that Siglec-15 from the plasma membrane did not contribute to its localization to lysosomes. Considering that Golgi transportation can mediate proteins to localize to lysosomes, we focused on the CI-MPR, a type-I integral membrane glycoprotein that binds to mannose 6-phosphate (M6P)-containing lysosomal proteins and transports them from the Golgi to the endosomal–lysosomal system (Coutinho et al., 2012; Ghosh et al., 2003). Ultra-high super-resolution microscopy analysis and Co-IP showed that CI-MPR and Siglec-15 were co-localized (Fig. 4A and 4B), and such co-localization occurred in the Golgi and endosomes (Fig. 4C). In line with this, BLI showed a direct interaction between Siglec-15 and CI-MPR (Fig. 4D). Low lysosomal pH triggers CI-MPR dissociation with cargo in the lysosomes and allows CI-MPR to recycle to the trans-Golgi network (TGN; Wang et al., 2020). Consistent with this, immunostaining showed minimal colocalization of Siglec-15 and CI-MPR in the lysosomes (Fig. 4C). Blocking this transport route with CI-MPR knockdown led to a decrease of Siglec-15 in the lysosomes (Figs. 4E and S4B–D), suggesting that CI-MPR mediates the transport of Siglec-15 from the Golgi to lysosomes. We then dissected the structural basis of the binding of Siglec-15 to CI-MPR. Structurally, Siglec-15 may contain mannose-6-phosphate owing to its N-linked glycosylation (Chen et al., 2020), prompting us to speculate that Siglec-15 binds to CI-MPR via M6P. Using a published method (Pechincha et al., 2022) to detect M6P-containing proteins, we verified that Siglec-15 contained M6P modifications (Fig. 4F). In addition, we found that treating tumor cells with tunicamycin, an inhibitor of N-linked glycosylation (Oliveras et al., 2020), disrupted the association between Siglec-15 and CI-MPR (Figs. 4G and S4E). Consistently, mutation of the Siglec-15 glycosylation site (N172Q) abrogated the interaction of Siglec-15 with CI-MPR in Daoy cells. In contrast, mutation of the sialic acid-binding site (R143A) did not generate a similar result (Fig. 4H). As expected, the N172Q mutation reduced Siglec-15 localization to the lysosomes, the release of Ca²⁺ from lysosomes, and cell viability (Fig. 4I and S4F–S4H). In addition, knockdown of CI-MPR counteracted the promoting effect of Siglec-15 on tumor growth in vivo (Fig. 4J). These results suggest that CI-MPR binds Siglec-15 and transports Siglec-15 from the Golgi apparatus to lysosomes.

Siglec-15 is upregulated by the transcription factor AhR

Next, we investigated the molecular basis of Siglec-15 upregulation in SHH-MB cells. By analyzing the possible transcription factor-binding sites, we found that the xenobiotic response element (XRE) with a core sequence (5′-GCGTG-3′) was present in the promoter region of Siglec-15, which is commonly recognized by AhR (Fig. 5A), a critical cytoplasmic transcription factor that senses xenobiotics and metabolites (Larigot et al., 2022). Previously, we showed that AhR profoundly regulates tumorigenic cell behaviors (Liu et al., 2017, 2018a, 2018b). Coincidentally, AhR has been reported to promote central nervous system tumor development (Opitz et al., 2011; Takenaka et al., 2019), suggesting that Siglec-15 may be regulated by AhR. ChIP-qPCR showed that AhR bound to the Siglec-15 promoter region, and its transactivation was verified by luciferase assay (Fig. 5B and 5C). Kynurenine (Kyn), a typical AhR ligand that is produced from indole-2,3-dioxygenase 1 (IDO1)-catalyzed tryptophan, was found to promote the entry of AhR into the nucleus and upregulate Siglec-15 expression (Figs. 5D, 5E and S5A). As expected, the AhR inhibitor StemRegenin 1 (SR1) or CH-223191 downregulated the expression of Siglec-15 as well as CYP1A1 and CYP1B1, two AhR-targeted genes (Fig. 5E and S5A). AhR knockout also downregulated Siglec-15 expression in Daoy cells (Figs. 5F and S5B–S5D). Similar results were obtained in primary SHH-MB tumor cells (Figs. 5D–F and S5C–E). In addition, we found that AhR levels in the nucleus were correlated with Siglec-15 expression in SHH-MB patients (Fig. 5G and 5H). In line with these results, Daoy cells highly expressed IDO1, leading to high levels of kynurenine (Kyn) in Daoy xenografts (Fig. 5I and 5J). IDO1 knockdown reduced Kyn levels and abrogated AhR activity; however, this effect was rescued by the addition of exogenous Kyn (Figs. 5L, 5M, S5G, S5I and S5J). Moreover, we found that the tryptophan transporter SLC1A5 was highly expressed in Daoy and primary SHH-MB cells, and knockdown of SLC1A5 also resulted in AhR inactivation, coupled with decreased Kyn levels (Figs. 5K–M, S5F and S5H–J). Together, these results suggest that AhR transcriptionally regulates Siglec-15 expression.

Targeting AhR effectively treats SHH-MB in vivo

Finally, we investigated whether targeting AhR could achieve therapeutic efficacy. To this end, we treated Daoy cells with the AhR inhibitor SR1 or CH-223191, and found that cell viability was markedly impaired (Fig. 6A). Such reduced cell viability was also observed in AhR-knockout Daoy cells (Fig. 6B), but was rescued by Siglec-15 overexpression (Fig. 6C). A similar result was obtained from primary tumor cells of SHH-patients (Fig. S6A and S6B). To validate these results in vivo, we established luciferase-expressing Daoy MB xenografts. Intravenous administration of SR1 or CH-223191 every 2 days, 1 week after intracranial injection (Fig. 6D), we found that AhR inhibition retarded tumor growth, as evidenced by bioluminescence radiance and H&E staining (Fig. 6E–G), concomitant with increased mouse weight and prolonged survival (Fig. 6H and 6I). In line with this, decreases in Siglec-15 expression and reduced entry of AhR into the nucleus were observed in the treated tumors (Fig. 6J). As expected, AhR knockout also resulted in inhibition of tumor growth (Fig. S6C–F). Intriguingly, forced overexpression of Siglec-15 counteracted the treatment effect of SR1 or CH-223191 on tumor growth in the brain (Fig. S6G and S6H). Together, these results suggest that targeting AhR results in the inhibition of SHH-MB growth by regulating Siglec-15 expression.

Discussion

Siglec-15 is thought to act as an immune checkpoint and a potential target for cancer immunotherapy. In this study, we unexpectedly revealed the tumor-promoting effect of Siglec-15 in SHH-MB beyond its immune regulation. We found that AhR, Siglec-15, and TRPML1 formed an axis that promoted SHH-MB development. Similar to common secretory proteins, Siglec-15 uses Golgi machinery for transport. However, the upregulation of Siglec-15 by activated AhR and the increased M6P modification resulted in CI-MPR-mediated translocation of Siglec-15 from the Golgi to lysosomal membranes, where Siglec-15 interacts with TRPML1 to initiate the release of lysosomal Ca²⁺ into the cytoplasm. This series of molecular events ultimately triggers the activation of Ca²⁺ signaling and the transcription factor TFEB, leading to the promotion of SHH-MB growth (Fig. 6K).

Mounting evidence has demonstrated that AhR is of paramount importance in cancer biology and immunology (Liu et al., 2017, 2018a; Takenaka et al., 2019). The maintenance of AhR activity in SHH-MB requires active tryptophan (Trp) metabolism. In the cytoplasm, Trp can be catalyzed by IDO1 into Kyn, an endogenous ligand that activates AhR. In line with this, IDO1 was highly expressed in SHH-MB malignant cells. The Trp transporter SLC1A5 was also highly expressed, thus replenishing intracellular tryptophan consumption via active transportation. As a critical cytosolic transcription factor, AhR acts as an exposome receptor that maintains cellular homeostasis by detoxification of xenobiotics (Suzuki et al., 2020). Previously, we found that activated AhR may induce tumor-repopulating cells (TRCs) into a dormant state (Liu et al., 2017). The underlying explanation is that cells use dormancy to counteract sublethal invaders and wake up after spontaneous decay of the invader within the cell. Contradictorily, in this study, we found that activated AhR promoted SHH-MB cell growth rather than dormancy. This may be explained by the fact that rapid cell growth and proliferation may also dilute toxins or invaders. The key question is, what is the switch point that guides cells to enter a dormant or proliferating state? AhR exerts its detoxifying function by transactivating cytochrome P450s (Suzuki et al., 2020). As heme-containing monooxygenases, P450 enzymes catalyze the incorporation of one oxygen atom from molecular oxygen into the toxic molecule RH, yielding innoxious ROH and reactive oxygen species (ROS) as byproducts. However, our recent studies have demonstrated that AhR acts as a ROS sensor by regulating glycogenolysis and the pentose phosphate pathway (Zhou et al., 2023). In the present study, we found that SHH-MB cells expressed higher levels of ROS than other tumor cell lines. This may explain how AhR selectively regulates Siglec-15 to promote SHH-MB growth. Consistently, it has been reported that elevated ROS levels can activate TRPML1, leading to calcium release and enhanced TFEB nuclear translocation (Zhang et al., 2016), providing further insights into the role of the Siglec-15 and TRPML1 combination in regulating TFEB and promoting tumor growth. A deeper understanding of the role of ROS in the AhR-Siglec-15-TRPML1-TFEB axis warrants further investigation.

Evidence further suggests that Siglec-15 is predominantly localized in the lysosome. As a metabolic signaling center, lysosomes possess the ability to detect the presence of nutrients and growth factors, thereby regulating cellular metabolism (Jewell et al., 2013; Li et al., 2019; Medina et al., 2015). In this study, we demonstrated that the interaction between Siglec-15 and TRPML1 leads to calcium release, subsequently activating TFEB. Given that lysosomes serve as an intracellular reservoir for calcium, they play a crucial role in maintaining Ca²⁺ homeostasis and signal transduction (Medina and Ballabio, 2015; Medina et al., 2015). Previous studies have pointed out that TFEB transcriptionally activates the lysosome autophagic pathway during starvation (Medina et al., 2015). Thus, our results provide a novel effect of TRPML1 release calcium trigger TFEB dephosphorylation and nuclear translocation. It is important to note that lysosomal proteins originate from two sources: endocytosis and the autophagic pathway (Saftig and Klumperman, 2009; Yu et al., 2018). The classic lysosomal localization label is mannose 6-phosphate (M6P) (Ghosh et al., 2003). This requires a specific sequence (Asn-X-Ser/Thr) in the protein, which is produced by continuous processing and modification of the ER and the Golgi apparatus (Bonifacino and Traub, 2003). Evidence showed that Siglec-15 is expressed not only on the cell membrane but also in the cytoplasm; however, blocking the endocytosis pathway of Siglec-15 does not affect its localization on the lysosome. In line with this, we demonstrated that Siglec-15 binds to CI-MPR through its N-glycosylation binding site N172. Both N172Q mutation and CI-MPR knockdown resulted in the reduction of Siglec-15 localization to the lysosome and of the promoting effect on tumor growth without the alteration of Siglec-15 on the plasma membrane, suggesting that the Siglec-15 on lysosomes rather than on the plasma membrane promotes cell proliferation. Therefore, inhibition of Siglec-15 lysosomal transport pathway can block MB development, which also provides a new idea for tumor-targeted therapy.

In summary, the data in the present study revealed that AhR activation promotes Siglec-15 transcription by binding to TRPML1 on the lysosome after being transported by CI-MPR. Siglec-15 promotes Ca²⁺ release, which leads to TFEB dephosphorylation and nuclear translocation, thereby promoting SHH-MB growth. The potential therapeutic implications of AhR-Siglec-15 axis modulation in MB cannot be overlooked. Targeted therapies may offer new avenues for treatment, either as standalone interventions or in combination with existing therapies.

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