ASPORIN: A root of the matter in tumors and their host environment

ASPN is also known as periodontal ligament-associated protein 1 (PLAP1) [ 3 ]. The name asporin (ASPN) refers to the existence of conserved aspartate (D) residues (6–19) in its N-terminal portion and sequence similarity (54%) to DCN [ 6 ]. The distinct length of D-residues in the N-terminus ranging from 12 to 16 D-repeats are causative for 10 different types of ASPN polymorphisms in cancer and bone-related diseases. While the functional significance and difference of such polymorphic ASPN need to be elucidated clearly, it is known to block collagen fibrillogenesis by competing with DCN in binding the same sites in collagen [ 41 ]. The D-rich N-terminal region of ASPN binds to type II collagen, while the central part, LRR10–12, binds to type I collagen [ 42 ]. This inhibits the collagen’s mineralization process and blocks the deposition of minerals such as calcium and phosphate in the collagen framework, which are essential in providing strength and support to cells of various body tissues, especially bones [ 3 ]. ASPN is majorly expressed by CAFs or reactive fibroblasts, also known as biologically active fibroblasts, and is crucial in tumor growth and progression. Stromal fibroblasts primarily express ASPN in various cancers, including pancreatic cancer (PC), prostate cancer (PCa), gastric cancer (GC), and colorectal cancer (CRC) [ 43 ]. On the contrary, ASPN has been observed to play a dual role in breast cancer (BC), i.e., displaying both pro- as well as anti-tumor effects [ 8 ]. ASPN is mostly known to regulate TGF-β, EGF, and CD44 signaling pathways in a TME and, therefore, can possess either pro- or anti-tumorigenic effects and has a tissue-specific role [ 4 ]. For example, the TGF-β/Smad2/3 signaling pathway is known to be activated by ASPN in CRC by interacting and inducing the translocation of Smad2/3 into the nucleus and facilitating the transcription of EMT-promoting genes [ 44 ]. On the other hand, ASPN derived from fibroblasts, i.e., CAFs in BC, inhibits the TGFβ1 receptor, thereby blocking TGFβ signaling pathway and suppressing tumor proliferation [ 45 ]. Further, ASPN has also been observed to activate pEGFR and its downstream effector pERK1/2 in GC [ 32 ]. Next, ASPN is observed to activate Rac1 via interacting with CD44 in both CAFs and nearby cancer cells in scirrhous GC. This allows cancer cells to become motile and migrate. ASPN is a distinct secretory protein that CAFs generate, encouraging the coordinated invasion of CAFs and cancer cells [ 46 ]. In this section, we will elaborately describe the role of ASPN and its associated signaling in various diseases ( Figure 2 ). The TGF-β family utilizes certain ways to transmit signals from outside of the cell’s environment to the intrinsic cytosolic region of the cell. For instance, they will be released as molecules (as ligands) attached to the ECM and processed before they can transmit the signals. These steps are crucial for adequately functioning of the TGF-β signaling process [ 47 ]. In cancer, the activation of TGF-β/Smad2/3 signaling via serine-threonine kinase can either act as a tumor promoter or tumor suppressor. It was reported that in BC, ASPN expression was promoted by TGF-β1 while inhibited by IL-1β in both normal breast fibroblasts and CAFs [ 45 ]. In the case of TNBC, ASPN acts as a tumor suppressor by interacting and blocking TGF-β1 and its following Smad2/3 activation that reduces EMT and stemness of cancer cells [ 45 ]. As both extracellular proteins, TGF-β1 and ASPN, are located around the cells in human articular cartilage, ASPN interacts physically with TGF-β1 via its LRRs and not through its conserved D residues. Hence, it was identified by affinity cross-linking experiment that ASPN, acting as an antagonist, inhibits the binding of TGF-β1 to its type 2 TGF-β receptor (TGF-β2) to form a dimer, a cell surface receptor, thereby blocking TGF-β signaling [ 14 ]. On the other hand, in CRC, intercellular ASPN acts as an oncoprotein by directly interacting with the downstream Smad2/3 and inducing the translocation of phosphorylated Smad2/3 (pSmad2/3) in the nucleus to facilitate the expression of oncogenes, leading to EMT and tumor progression [ 44 ]. Therefore, in TGF-β/Smad2/3 signaling, the interacting partners of ASPN, as well as its localization, decide the role of being a tumor suppressor or tumor promoter. In OA, ASPN inhibits TGF-β/Smad2/3 signaling, inhibiting AGC1 and COL2A1 expression, ultimately promoting the disease [ 28 ]. EGFR belongs to the receptor tyrosine kinase (RTK) family, which is observed to be highly upregulated in cancers such as glioblastoma (GBM), non-small-cell lung cancer (NCSLC), metastatic CRC, PC, BC, and head and neck cancers. Many mutations and truncations to the extracellular domain of EGFR lead to its overexpression in cancers such as EGFRvIII truncations, mutations in the kinase domains such as L858R, T790M, and exon 19 truncations. Further, EGFR overexpression induces other downstream pro-oncogenic pathways, such as RAS-RAF-MEK-ERK-MAPK and AKT-PI3K-mTOR, that leads to tumor proliferation and poor prognosis [ 48 ]. According to Wu, et. al. the CRC cell lines such as HT-29 and LoVo exhibited increased p-EGFR Tyr1173 and p-Src Tyr416 upon ASPN overexpression, whereas, on ASPN knockdown, EGFR Tyr1173 and p-Src Tyr416 levels were decreased. Further, p-EGFR and p-Src promoted the phosphorylation of cortactin (p-cortactin Tyr421 ). Cortactin tyrosine phosphorylation is crucial for effective ECM disintegration and metastasis in vivo , as well as to produce free actin barbed ends necessary for actin polymerization in invadopodia [ 49 ]. Similarly, in GC, ASPN plays an oncogenic role by promoting tumorigenesis and metastasis via influencing the EGFR and further ERK-CD44/MMP-2 signaling pathway [ 32 ]. Furthermore, Zhan et al. demonstrated that ASPN interacts with HER2 to activate HER2 signaling mechanism to promote thyroid tumor metastasis via regulating EMT phenotype through MAPK pathway [ 50 ]. CD44 is a non-kinase, cell surface transmembrane glycoprotein a widely known cancer stem cell (CSC) marker that undergoes alternative splicing in CSCs to promote cancer growth. CD44 and its various isoforms significantly promote PC, PCa, BC, CRC, and head and neck squamous cell cancers [ 51 ]. It belongs to the family of cell adhesion molecules (CAMs), which is crucial for cellular adherence to the ECM and cell communication [ 52 ]. In most cancers, the upregulation of CD44 promoted CSC traits such as self-renewability, EMT, and chemo- and radio-therapy resistance, which ultimately drives tumor proliferation, metastasis, invasion, migration, and stemness. The common ligands for CD44 are hyaluronic acid (HA), which is an important ECM component [ 52 ], osteopontin (OPN), and matrix metalloproteinases (MMPs), which are associated with various cancer-related signalings [ 53 ]. In PC, ASPN is significantly expressed by pancreatic stellate cells (PSCs), which interact with CD44 on pancreatic cancer cells (PCCs), activating the NF-κB/p65 pathway to induce EMT in PCCs. Also, AKT and ERK signalings are involved in driving the NF-κB/p65 pathway in PC, which can be in both autocrine and paracrine manner [ 54 ]. Furthermore, scirrhous gastric cancer has the worst prognosis among the GCs, which expresses ASPN through its CAFs. The ASPN interacts with CD44 to activate Rac1, which induces cancer cell invasion by CAFs via the paracrine mechanism [ 46 ].

The most common chronic joint disease, osteoarthritis (OA), affects the knees, hands, hips, and spine. It is the most common musculoskeletal cause of reduced mobility in older people worldwide. Several risk factors have been identified for OA, including age, genetic susceptibility, obesity, and joint misalignment [ 114 ]. When compared to healthy cartilage, it was shown that OA had higher levels of ASPN expression. The mRNA and protein expression levels of ASPN were elevated in peripheral blood samples from OA patients, indicating that it may be employed as a biomarker for OA diagnosis and tracking the disease’s development. ASPN’s capacity to bind collagens is an important property. The D-rich N-terminal region and core portion of the ASPN molecule bind to type II collagen, whereas the central region, LRRs 10– 12, binds to type I collagen. In vitro , ASPN dramatically reduced collagen fibrillogenesis in a dose-dependent manner. ASPN competes with DCN for binding to the same sites, and this competition in binding may play a role in controlling ECM growth. Polymorphisms in the calcium-binding site of ASPN, i.e. the N-terminal poly-D domain, are closely related to OA and PCa [ 3 ]. Additional data from human clinical trials may strengthen the connection between the D-repeat polymorphism of ASPN and susceptibility to OA [ 115 ]. As mentioned earlier, ASPN functions as a TGF-β negative regulator, according to in vitro findings. Recently, it was identified that ASPN promoted chondrocyte senescence and amplified cartilage impairment by blocking the TGFβ1-Smad2 pathway. Also, ASPN mRNA was identified to be a direct target of miR-26b-5p, which plays an important role in inflammation regulation, and its expression decreased in early OA compared to late OA. Hence, miR-26b-5p-ASPN-Smad2 axis can be a potential biomarker and therapeutic target in OA [ 116 ]. According to Rosas et al. there was an increased metastatic disease in individuals with OA. Additionally, prostate tissue and perhaps far-off lymphatic nodes showed elevated cartilage oligomeric matrix protein (COMP) levels in individuals with OA [ 117 ]. Furthermore, research suggests that joint replacement surgery may reduce OA’s capacity to encourage metastasis, influencing cancer treatment plans and survival rates for the most frequent cancer-related mortality, especially metastasis. The 14 D residues in the D14 polymorphism have been identified as an OA risk allele. ASPN has also been linked to disc degeneration and has been implicated in the development of OA. A meta-analysis revealed that Chinese and Japanese people with the D14 allele had a greater risk of lumber disc degeneration. It has been shown that the expression of the protein ASPN in vertebral discs rises with disc degeneration. In human nucleus pulposus cells, IL-1 enhanced the production of ASPN by activating the p65 pathway. Additionally, by attaching to the 41/31 bp region of the ASPN promoter, p65 mediated ASPN expression. ASPN served as a functional intermediary between IL-1 inhibition of aggrecan and collagen expression and TGF-induced aggrecan and collagen production in human nucleus pulposus cells [ 118 ]. Polycystic ovary syndrome (PCOS) involves the enlargement and loss of function of ovaries, high androgen levels, and insulin resistance. It is a heterogenous endocrine disease that affects women of reproductive age globally [ 119 ]. The intricate process of steroidogenesis, which results in androgen production in theca cells (TCs), involves a variety of biochemical indicators. Numerous triggering factors, including stem cell factor, IGF-1 inhibitor, and proteins with inhibitory effects, such as TGF-β, BMP, and activin, are involved in this process. For these reasons, the female reproductive system will be impacted by any disruption in the TCs’ normal operation [ 120 ]. ASPN is reported to be released by TC/interstitial cells in mouse ovaries and is a useful marker for secondary follicle formation at the gonadotropin-independent stage. This investigation discovered that ASPN inhibits the TGF-β/SMAD2–3 cascade and hypothesized that ASPN may have an autocrine/paracrine function in folliculogenesis [ 121 ]. Additionally, ASPN could influence testosterone synthesis via similar mechanisms. Further, it was shown that patients with PCOS had considerably increased serum ASPN levels, which may be related to the development of this condition. The androgen pathway may be responsible for this connection [ 120 ].

Mouse models are vital for investigating diseases associated with specific genes and proteins and for optimizing targeted therapies against specific diseases. Maccarana et al. generated ASPN knockout mice to evaluate the coherent changes associated with ECM composition, structural, biochemical, and biomechanical functions and properties of the skin. They found an overall upregulation of specific collagen genes (Col1α1, Col1 α 2, Col3 α 1) but no changes in overall collagen protein content, lysyl oxidases (Lox, Loxl2), and matrix metalloproteases (Mmp2 and Mmp3). In addition, the other SLRPs, BGN, and DCN and chondroitin sulfate/dermatan sulfate (CS/DS) content is doubled because of targeted disruption of exon 2–3 of ASPN −/− in skin tissues [ 122 ]. Similarly, the CS/DS content was increased in skin DCN knockout mice (Dcn −/− ) mice [ 123 ]. These studies offer a mechanistic explanation of SLRP deficiency and its associated ECM modulation. Thomson’s group has demonstrated that ASPN can be regulated through p53 in a PCa mouse model. Through immunohistochemistry, they confirmed the stromal expression of ASPN in conditional p53 and Rb knockout mouse models [ 60 ]. However, validation using Si/ShRNA studies targeting ASPN is required to confirm the loss of p53 and /or Rb loss and its associated expression of ASPN in the stroma. In an orthotopic mouse model of PCa, stable overexpression of D13 and D14 variants of ASPN in fibroblast cells does not show any variation in the xenograft weight but exhibits a difference in the incidence of metastasis. The D14 variant introduced xenografts displayed increased metastasis and dissemination to lymph nodes, liver, lung, and pancreas, whereas mice with D13 xenograft showed significantly less metastasis and specifically presented micrometastasis [ 33 ]. This study demonstrated the variant-specific role of ASPN in mouse models with more relevance to biological and metastasis phenotype. Recently, Hurley and colleagues demonstrated that ASPN is a secretory factor involved in PCa metastasis. They tested subcutaneous implantation of metastatic PCa cell lines derived from Hi-Myc mouse into ASPN WildType (WT) and ASPN knockout (KO) mice. Similar to the D13 and D14 orthotopic mouse models, there is no variation in the growth of subcutaneous allograft tumors between ASPN WT , ASPN homo, and heterozygous KO mice. Nevertheless, allograft tumors grown in AAPN homo KO mice had significantly low MSCs, CD44 positive CSCs, and increased tumor infiltrating CD8 + T cells compared to ASPN WT mice with more (67%) incidence of lung metastasis [ 59 ]. Thus, all these studies highlight the role of ASPN as a major factor in the TME influencing multiple cellular phenotypes, such as MSC, CSC, and immune cells, ultimately supporting distant metastasis.

Polymorphisms in the D-repeats of the gene encoding ASPN are first identified in osteoarthritis (OA) patients [ 28 ]. OA is considered the most common arthritis form in humans. ASPN is observed to be highly expressed in the articular cartilage of OA patients. A triplet repeat found in exon 2 of the ASPN gene codes for a polymorphic stretch of D-residues in the protein’s N-terminal region. There are 10 alleles, each encoding 10–19 residues in this repeat polymorphism (D-repeat), and the D13 allele is the most prevalent [ 29 ]. A meta-analysis by Wang et al. reported that ASPN D13 repeat polymorphism is associated with low risk for OA in Caucasian males. In contrast, D14 polymorphism is critical in male patients with Knee OA (KOA) [ 30 ]. Similar results of the D14 allele of ASPN and its association with KOA are also observed in the Mexican mestizo patient population [ 31 ]. The D14 allele of the ASPN gene is detected to be over-represented compared to the D13 allele, most commonly in knee and hip arthritis, and this frequency increases with the disease’s lethality. Furthermore, ASPN represses the gene expression of aggrecan (AGC1) and type II collagen (COL2A1) and inhibits proteoglycan accumulation in chondrogenesis by inhibiting TGF-β. The higher inhibition of TGF-β is associated with the higher D14 allele in the ASPN gene [ 28 ]. Specifically, Kou et al. found that ASPN protein (amino acid 159–205) interacts with TGFβ1 and represses its corresponding downstream genes associated with the cartilage matrix. These reports suggest that the effect of ASPN on TGF-β is allele-specific [ 32 ]. In addition, D13 and D14 ASPN polymorphisms were also found to be associated with developmental dysplasia of the hip (DDH), which later could develop into hip OA [ 29 ]. The unique stretch of D-repeat in ASPN N-terminal region leads to the germline polymorphisms in this D-repeat stretch, further resulting in degenerative disorders and are causative for driving cancer metastasis [ 33 ]. Orr et. al. observed an increased ASPN expression in the cancer-associated fibroblasts (CAFs) from prostate cancer (PCa) tumor microenvironment (TME) [ 34 ]. More than 10 D-repeat polymorphisms are observed in ASPN, commonly occurring between 12–16 D-residues. These polymorphisms drive several disorders such as OA by blocking chondrogenesis [ 28 ], lumbar-disc degeneration (LDD) [ 35 ] and ankylosing spondylitis [ 36 ]. These D-repeat polymorphisms inhibit BMP-2 and TGF-β signaling that promotes bone lesions and metastasis in PCa [ 37 – 39 ]. A study conducted by Hurley et. al. in men who underwent radical prostatectomy (RP) found that the ASPN D-repeat lengths ranged from 10 to 19 D-residues, with 90% of the men having alleles comprising 13, 14, or 15 residues. The most prevalent ASPN D-length genotypes in men with PCa were 13/15, 13/13, 15/15, 14/15, and 13/14, with the other genotypes accounting for 0.1% to 5.8% in the study. They discovered through Cox regression analysis that germline ASPN D13/14 was substantially linked to post-surgery metastatic recurrence. Multivariable studies also revealed that compared to all other alleles, germline bearers of the ASPN D14 allele were considerably more likely to develop metastasis. Hence, hereditary ASPN D13/14 or any ASPN D14 may increase the chance of metastatic recurrence after surgery [ 33 ]. Recently, cancer-associated ASPN mutations have been identified in colon cancer. Based on the DNA mismatch repair (MMR) profile between cancer and normal cells isolated from colon cancer, the researchers identified rare c.198delT (p.Pro67HisfsX24, 1 in 100 tumors analyzed) and c.198dupT (p.Pro67SerfsX3, 2 in 100 tumors analyzed) mutations in ASPN [ 40 ]. ASPN is also predicted to have both tumor suppressor and promoter roles in colon cancer apart from TNBC.

The bone is the third leading site for cancer cell metastasis after the lung and liver [ 109 ] because of its composition, especially its ECM, high metabolic state due to constant turnover, and growth signaling pathways that support cancer cells to grow and survive. The bone ECM comprises of different cell types, such as osteoblasts, osteoclasts, and osteocytes, which are embedded within the organic and inorganic components [ 110 ]. The proliferation of cancer to metastasis is not just carried by cancer cells alone but involves the participation of different cell types in a TME. The MSCs secrete ASPN, which inhibits BMP-4-mediated lineage differentiation of MSCs by being bound to BMP-4 and enhances MSCs self-renewability, thereby causing metastatic development [ 59 ]. Hughes et al. observed the reduction in MSCs stem cells and an increase in CD8 + T cells in ASPN-null mice carrying PCa. This significantly reduced the lung metastasis [ 59 ]. MSCs, on lineage differentiation, form physiologic cell lineages such as osteoblast (an important component of bone ECM), adipocytes, and chondrocytes [ 102 ], which ASPN inhibits. Hughes et al. observed that both bone marrow-derived and tissue-resident human and mouse MSCs exhibit significant levels of ASPN expression [ 59 ]. The bone matrix is composed of cytokines and growth factors that include the TGF-β family involving BMP-2, BMP-4, and TGF-β1, which play a crucial role in the differentiation of MSCs to form osteoblast [ 111 , 112 ], and ASPN is a known antagonist for these factors. Ming et al. demonstrated that in TGF-β receptor 2 (TGF-βR2) knockout in osteoblasts of mice of PCa bone metastasis, there was an increase in bone lesion with the upregulation of basic fibroblast growth factor (bFGF), which acts as a mediator of PCa growth. Therefore, as explained earlier, TGF-β signaling is used by cancer cells to grow and proliferate, whereas, in the case of bone metastasis, the loss of TGF-β signaling in osteoblasts induces bFGF, which promotes bone metastasis in PCa [ 113 ]. Therefore, ASPN, being secreted from MSCs, inhibits its lineage differentiation by blocking BMP-4 and a known inhibitor of the TGF-β signaling, can promote bone metastasis by interacting with TGF- βR1 and blocking its dimerization with TGF-βR2 and ultimately dysregulating the TGF-β signaling pathway.

ASPN is one of the major SLRP recently getting attention in various cancers and other diseases. SLRPs contain 18 members, which are classified into 5 classes based on chromosomal arrangements and homology at the gene and protein levels. The amino acid sequence of ASPN is 50–70% identical to that of DCN and BGN, but it varies from these two in that it has a unique D-repeat stretch in the N-terminal region [ 124 ]. D-repeats range from D9 to D20, and each cancer/disease varies with a unique number of D-repeats that may have a unique function in the pathogenesis of OA and cancer. Hence, it is essential to elucidate the germline variation in ASPN (homozygous and heterozygous alleles) and its association among various cancer progression, metastasis, and clinical outcomes. The majority of studies on ASPN have demonstrated its extracellular or secretory protein function from the CAFs, which is essential to influence the cell membrane receptors’ activation or repression of the signaling, thereby influencing tumor growth and proliferation. However, the significance of its intercellular localization in the cytoplasm or nucleus, its interacting partners, and the downstream effector proteins are not properly elucidated. In the cancer context, ASPN expression needs to be investigated stage-wise. For instance, ASPN is enriched in low-grade glioma tumor endothelial cells with tumor-suppressing function. In contrast, high-grade glioma does not show such an effect [ 125 ], demonstrating ASPN functional heterogeneity among different tumor grades. The dual role of tumor suppressor versus tumor promoter function of ASPN still needs to be fully elucidated. In the future, the subtype-specific role and function of ASPN can be determined using a large patient population through multicentered clinical investigation identifying high or low ASPN expression and corresponding clinical correlation. Overexpression of ASPN has been associated with oxidative stress in GC cells [ 43 ]. In this context, we must explore the drug or radiation therapy-induced enrichment or overexpression of ASPN and its association with the induction of oxidative stress or resistance. Recent studies have provided expression patterns of ASPN in stromal versus epithelial compartment, but the role of ASPN in tumor heterogeneity and CAF heterogeneity remains unexplored. Tumor stroma also contains immune cells, which were shown to be modulated in the bladder, prostate, and endometriosis [ 59 , 126 , 127 ]. However, a major drawback in previous research is that most of the data were gene correlative studies between ASPN and immune markers extracted from publicly available data sets. The relationship between ASPN and immunomodulatory/stimulatory factors needs to be investigated through in vitro and in vivo models exploring high ASPN expression versus immune pathways and immune suppression in detail in various cancers. To date, siRNA targeting ASPN in keloids, a proliferative disorder of fibroblasts, showed promising results in controlling fibroblast growth and keloid metabolism [ 128 ]. Moreover, the co-delivery of siRNA or shRNA targeting ASPN enhanced oxaliplatin efficiency and helped to overcome oxaliplatin-mediated chemoresistance in CRC cells [ 129 ]. Hence, pharmacological inhibitors targeting ASPN directly or indirectly interfering with the binding of ASPN with TGFβ, NFκB, BMP2, BMP4, CD44, EGFR, and HER2 signaling is urgently required as ASPN gene targeting approach in patients have some limitations. Recently, some drugs targeting specific molecules have been explored through connectivity mapping. Similarly, ASPN-targeting drugs must be identified using cancer-specific gene signatures (TCGA or GEO) to repurpose. All these compounds need to be tested in in vitro cell lines, tumoroid, and spontaneous mouse models with different genetic backgrounds to identify potential candidates for ASPN-targeted therapies. CRISPR KO ASPN mice need to be developed and crossed with respective organ specific Cre to explore the impact of ASPN loss and its associated non-epithelial cell crosstalk in specific cancers. ASPN targeting has emerged as a vital strategy to overcome lung fibrosis, and targeting ASPN has been viewed as a beneficial factor for cardiac remodelling [ 130 ]. He and colleagues recently reviewed the role of ASPN in regulating TGFβ Receptor 1 recycling and activation of the SMAD pathway in lung fibrogenesis [ 131 ]. Hence, it will be imperative to identify a peptide blocker as an inhibitor to prevent primary lung fibrosis. This approach can be translated to cancer with predominantly activated stroma, as in the case of pancreatic cancer. Overall, ASPN is a unique target in both tumors and fibroblasts to reduce disease burden and improve the survival of millions of cancer patients or bone-related diseases.

SLRPs are a complex subgroup of proteoglycans that are known to be involved in matrix structure, cell development, and cellular signaling. They are distributed across the ECM. They comprise 17 members, further divided into 5 classes based on chromosomal arrangement, LRRs, cysteine-rich clusters at the N-terminal, and evolutionary protein conservation [ 9 ]. The SLRPs classification, molecules-associated with each class with their description of their biological functions, and associated diseases are briefly described in Table 1 . Class I SLRP includes DCN, BGN, ECM2, and ASPN. DCN consists of a conserved protein core, 1- Glycosaminoglycan (GAG) side chain, and a central domain with 10-LRRs [ 10 ]. BGN has LRRs with 1 or 2 covalently attached GAG side chains. The N-terminus of the core protein has amino acid residues connected to tissue-specific chondroitin or dermatan-sulfate GAG chains [ 11 ]. ECM2 is a 699-amino acid peptide with an ORF of 2097 nucleotides and is encoded by the cDNA for ECM2. It displays similarity to several well-known ECM proteins, including proteoglycans, DCN, and KERA. The amino acid sequence includes many potential functional domains, such as an arginine-glycine-aspartic acid (RGD) sequence, a von Willebrand factor (VWFC) domain, and a LRR motif [ 12 ]. ASPN is expressed in cancer, periodontal ligaments, and cartilage cells/tissues. Due to sequence homology, its functions are partly correlated with DCN (54%) and BGN (60%). Basic differences exist in the molecular weights of these 3 class I SLRPs with ASPN: 42KDa, DCN: 36KDa, and BGN: 38KDa [ 13 ]. The relationship between ASPN, DCN, and BGN was discovered by cloning human and mouse ASPN cDNAs [ 6 ]. ASPN was primarily found in the skeleton and specialized connective tissues during embryonic development compared to major parenchymal organs in mice. Specifically, it is expressed in the periosteum, vertebrae, maxilla, mandible, and long bones [ 5 , 14 ]. The murine ASPN gene has 8 exons and spans on chromosome 13, whereas in humans, ASPN is located on chromosome 9q22.31 with 8 exons (26 kb), similar to that of BGN (7 kb, Xq27) and DCN (45 kb, 12q13.2) [ 3 , 5 ]. During human embryonic development, ASPN is expressed in the heart, aorta, liver, uterus, periodontal ligaments, dental follicles, and articular cartilage [ 5 , 7 ]. The ASPN gene is an intrinsic part of the SLRP family of gene clusters encoding osteoadherin, osteoglycin/mimecan, and extracellular matrix protein-2 (ECM2) [ 5 ]. The ASPN protein comprises 380 amino acids (aa) containing a 14-aa signal peptide, 18-aa pro-peptide, 4 amino-terminal cysteines, 10 LRRs, and 2 C-terminal cysteines. The asparagine 281 residue contains an N-glycosylation site with an O-glycosylation site at serine 54 [ 14 ]. ASPN lacks chondroitin and dermatan sulphate chains, which makes it different from DCN and BGN. Instead, it contains 8 to 19 (typically 13) D-repeats. Also, ASPN is not an absolute proteoglycan as it does not have numerous glycosylation sites such as serine/glycine dipeptide sequence for glycan attachment. On the other hand, DCN and BGN are immensely rich in polysaccharides and hence are considered genuine proteoglycans [ 3 ] ( Figure 1 ). Fibromodulin (FMOD), lumican (LUM), prolin and arginine-rich end leucine-rich repeat protein (PRELP), keratocan (KERA), and osteomodulin (OMD) or osteoadherin (OSAD) belong to class II SLRP. FMOD lacks chondroitin/dermatan sulfate chains. Instead, it contains N-linked keratan sulfate chains connected to many O-sulfated tyrosine residues in the N-terminal and numerous aspartic and glutamic acid residues. It also has conserved cysteine residues at both the N- and C-termini that form disulfide bonds to maintain the structural integrity of the core protein [ 15 ]. LUM has 4 distinct regions, including a 16-amino acid peptide, a negatively charged N-terminal region with tyrosine sulfate and disulfide bonds, a 6–10 LRRs motif with a typical molecular architecture that facilitates protein interactions, and a C-terminal region with 2- conserved cysteine residues [ 16 ]. PRELP has 382-amino acid residues, including a signal peptide of 20 residues. It lacks GAG chains and has a distinctive and conserved N-terminal domain that binds heparin and heparan sulfate in rodents, bovine, and humans [ 17 ]. KERA has an N-terminal signal peptide, which is followed by a highly conserved region with a 10-LRRs motif. There are 352 amino acids in the predicted protein. It resembles to LUM (41% amino acids identity) among the SLRP family members [ 18 ]. OMD or OSAD is a protein whose core is made up of 11 B-type LRRs that range in length from 20 to 30 residues. 4-potential tyrosine sulfation sites can be found in the whole main sequence, 30 of which are located at the N-terminal end of the molecule. There are 6 places where N-linked glycosylation might occur. It is the only known protein with a significant and very acidic C-terminal domain [ 19 ]. Class III SLRPs consist of epiphycan (EPYC) or dermatan sulfate proteoglycan 3 (DSPG3), opticin (OPTC), and osteoglycin (OGTC). In contrast to other SLRPs, EPY from salmon nasal cartilage has a GAG domain in the N-terminal. The 55 possible GAG modification sites (Ser-Gly and Gly-Ser) inside the GAG domain are all heterogeneously changed by GAG chains [ 20 ]. With almost 73% similarity in amino acid sequences, the LRR domains of the proteins OPTC, EPYC, and OGTC are homologous. Their amino-terminal regions, however, show notable variations. A collection of sialylated O-linked oligosaccharides is found in the amino-terminal region of OPTC [ 21 ]. OGTC contains multiple glycosylation sites, a distinctive cysteine-rich region sequence, and 6 LRRs. It can undergo controlled glycosylation. Further, adding GAGs and glycans to proteins in the endoplasmic reticulum and Golgi apparatus enables diverse functional roles [ 22 ]. The last two SLRP classes are non-canonical. Chondroadherin (CHAD), Nyctalopin (NYX), and Tsukushi (TSKU) fall under class IV SLRPs. CHAD has 10 LRRs and lacks an N-terminal extension but possesses two cysteine loops in its C-terminal domain. It contains xylose, galactose, mannose, and fucose, indicating the presence of short oligosaccharides with a linking structure similar to CS/DS GAG chains. However, it does not have the repetitive disaccharide characteristic of GAGs, as it lacks hexosamines [ 23 ]. NYX is a 481-amino acid protein with an N-terminal signal sequence, 11 central LRRs, and both N-terminal and C-terminal cysteine-rich LRRs. A linker region and a predicted GPI-anchorage site are succeeded by a C-terminal cysteine-rich LRR [ 24 ]. TSKU has LRRs flanked by conserved cysteine residues, but its C-terminus resembles category IV proteins, such as CHAD and NYX. The human TSK cDNA encodes a 337 amino acid-secreted protein, probably glycosylated, containing 9 LRRs and a 16 amino acid signal region. Finally, Class V SLRPs involve podocan (PODN) and podocan-like protein 1 (PODNL1) [ 25 ]. PODN is a 611-amino acid secretory protein with 20 LRRs. It has a distinctive N-terminal cysteine-rich cluster pattern and a highly acidic C-terminal domain [ 26 ]. PODNL1 comprises a signal peptide, a distinctive cysteine-rich N-terminal cluster, 21b LRR motifs, and a potential N-glycosylation site. It is structurally similar to PODN [ 27 ].

The extracellular matrix (ECM) is a non-cellular component that provides three-dimensional structure, physical scaffolding, and biochemical support to the surrounding cells. In a normal cell, ECM plays a critical role in cellular functions such as growth, differentiation into different cell types, survival, maintaining homeostasis (deposition, remodeling, and degradation), morphogenesis, and migration [ 1 , 2 ]. ECM comprises of macromolecules such as glycoproteins (mucins), proteoglycans, collagens, laminins, fibronectin, elastin fibers, and reticulin. Among these components, proteoglycans, glycoproteins, and collagens are regarded as “core-matrisome” of ECM. Small leucine-rich proteoglycans (SLRPs) are the major non-collagen component of ECM, playing a vital role in cancer progression, metastasis, cardiac remodeling, osteoarthritis (OA), tissue morphogenesis, and immune response. SLRPs consist of 17 members, classified into V classes based on chromosomal arrangements and homology at the gene and protein levels. Classes I to III are known as traditional or canonical classes, while IV and V are referred to as non-canonical classes. The SLRP family’s characteristic features include core leucine-rich repeats (LRRs), N-terminally concentrated cysteine-rich clusters (CX 3 CXCX 6 C), and C-terminal ear repeat motifs. In addition to having a common protein core, SLRPs include several linked glycosaminoglycan chains, including keratan, chondroitin, dermatan sulfate, and heparan sulfate. The polarized structure of SLRPs provides them their characteristic crescent shape, with β-sheets aligned on the concave surface and helices forming the convex face [ 3 ]. Class I SLRPs, which include decorin (DCN), biglycan (BGN), and asporin (ASPN), have the highest homology based on their amino acid sequence. DCN is regarded as a tumor suppressor by exerting anti-proliferative, anti-metastatic, and angio-suppressive effects. BGN acts as a tumor promoter by suppressing immune responses and supporting tumor progression. On the other hand, ASPN possesses both positive and negative roles in tumor progression in prostate, breast, colon, gastric, and pancreatic cancer while suppressing triple-negative breast cancer (TNBC) [ 4 ]. ASPN is localized in the ECM of several tissues, including collagen-enriched tissues such as cartilage and bones. Three different research groups discovered it in 2001 [ 5 – 8 ]. Apart from cancer, ASPN has a significant role in bone and joint diseases such as OA, hypochondrogenesis, and intervertebral disc disease [ 3 ].