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

Abstract

Asporin (ASPN) has been identified as one of the members of the class I small leucine-rich proteoglycans (SLRPs) family in the extracellular matrix (ECM). It is involved in classic ensigns of cancers such as self-dependent growth, resistance to growth inhibitors, restricting apoptosis, cancer metastasis, and bone-related disorders. ASPN is different from other members of SLRPs, such as decorin (DCN) and biglycan (BGN), in a way that it contains a distinctive length of aspartate (D) residues in the amino (N) -terminal region. These D-repeats residues possess germline polymorphisms and are identified to be linked with cancer progression and osteoarthritis (OA). The polyaspartate stretch in the N-terminal region of the protein and its resemblance to DCN are the reasons it is called asporin. In this review, we comprehensively summarized and updated the dual role of ASPN in various malignancies, its structure in mice and humans, variants, mutations, cancer-associated signalings and functions, the relationship between ASPN and cancer-epithelial, stromal fibroblast crosstalk, immune cells and immunosuppression in cancer and other diseases. In cancer and other bone-related diseases, ASPN is identified to be regulating various signaling pathways such as TGFβ, Wnt/β-catenin, notch, hedgehog, EGFR, HER2, and CD44-mediated Rac1. These pathways promote several cancer cell invasion, proliferation, and migration by mediating the epithelial-to-mesenchymal transition (EMT) process. Finally, we discussed mouse models mimicking ASPN in vivo function in cancers and the probability of therapeutic targeting of ASPN in cancer cells, fibrosis, and other bone-related diseases.

1. Introduction

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 (CX₃CXCX₆C), 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].

2. Structural characterization of SLRP family

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.

Table 1.

Classification of SLRPs and their associated functions and diseases

SLRPs	Classes	Members	Mol. Wt.	Function	Associated Diseases	Reference
Canonical	I	Decorin (DCN)	40 kDa	Regulates collagen fibrillogenesis, suppresses tumour metastasis and angiogenesis by blocking various RTKs.	Congenital stromal dystrophy (CSCD), cancer, glaucoma and duchenne muscular dystrophy (DMD)	[10, 132–134]
		Biglycan (BGN)	42 kDa	Interacts with innate immune receptors (TLR-2, -4) to induce inflammatory response leads to NF-κB supporting cancer growth and migration.	Atherosclerosis, cancer, DMD and rheumatic arthritis	[9, 134–136]
		Asporin (ASPN)	42 kDa	Secreted form blocks TGFβ signalling in OA and suppresses tumour whereas cellular form promotes TGFβ signalling by upregulating the Smad2/3 translocation to the nucleus, EGFR and CD44 signallings, causing tumour growth.	Cancer, endometriosis, ischemic cardiomyopathy, OA, rheumatoid arthritis, lumbar disc	[4, 14, 127, 130, 137]
		Extracellular matrix 2 (ECM2)	48 kDa	Regulates immune system, cell proliferation and differentiation supporting tumour growth and migration.	Glioma, idiopathic pulmonary fibrosis, pulmonary artery hypertension	[138, 139]
	II	Fibromodulin (FMOD)	40 kDa	Regulates collagen cross-linking, packing, assembly, and fibril architecture and functions as a ligand of many cytokines and growth factors, especially TGF superfamily.	OA, tendinopathy, atherosclerosis, heart failure, ulcerative colitis, skin fibrosis	[15, 140–142]
		Lumican (LUM)	38 kDa	Organizes tissues structurally such as bone matrix, facilitates EMT, cellular proliferation, migration, invasion, and adhesion.	Cornea dystrophy, cataract, chorioretinal diseases, bowen disease, actinic keratosis	[143–145]
		Proline and arginine-rich end leucine-rich repeat protein (PRELP)	55 kDa	Interacts with collagen types I and II, heparin, and heparan sulphate and possibly functions as an anchoring molecule for basement membranes to connective tissue.	Chronic lymphocytic leukemia Hutchinson-Gilford progeria (HGP) Hepatocellular carcinoma	[146–148]
		Keratocan (KERA)	41 kDa	Critical for corneal morphogenesis during embryonic development, maintain corneal topography in adults, regulates ECM synthesis by binding collagen fibrils with a protein moiety and extending highly charged GAG chains.	Keratoconus and cornea plana	[149–151]
		Osteomodulin (OMD) or Osteoadherin (OSAD)	49.116 kDa	Binds hydroxyapatite, regulates the structure of collagen fibrils, plays a significant role in bone disorders such OA and heterotopic ossification as well as biological processes like cell adhesion and tooth formation.	Diabetes mellitus, chronic kidney disease and atherosclerosis	[152, 153]
	III	Epiphycan (EPYC) or Dermatan sulfate proteoglycan 3 (DSPG3)	36 kDa	Provides GAG attachment sites to interact with other ECM proteins and collagen fibers for the production of type I collagen fibers, affects ECM organization, correlated with MMP, CXCL, and TGF-β expression.	Metastatic ovarian cancer, hereditary hearing loss	[154, 155]
		Opticin (OPTC)	90 kDa	Prevents angiogenesis by interfering with integrin-mediated interactions between endothelial cells and collagen.	Diabetic retinopathy, glaucoma	[156, 157]
		Osteoglycin (OGTC)	37 kDa	interacts with molecular targets in the ECM and influences processes like ECM assembly, immunity, corneal transparency, fibrosis, and cancer biology.	Chronic kidney disease, fibrosis, myocardial infarction, rheumatoid arthritis.	[22, 158, 159]
Non-canonical	IV	Chondroadherin (CHAD)	40.5 kDa	Regulates cytoskeletal organization of chondrocytes interacting with one another to facilitate cell adhesion.	Leiomyosarcoma, intervertebral disc degeneration	[23, 160, 161]
		Nyctalopin (NYX)	52 kDa	Interacts with TRPM1, a non-specific cation channel on dendritic tips of depolarizing bipolar cells (DBCs) in the retina.	Congenital stationary night blindness Åland island eye disease (AIED)	[162–164]
		Tsukushi (TSKU)	37.8 kDa	Vital role in various cell signalling pathways such as BMP, FGF, TGF-β, and Wnt.	Non-alcoholic steatohepatitis, lung cancer, neuroblastoma and hydrocephalus disease	[165, 166]
	V	Podocan (PODN)	67 kDa	Acts as a strong inhibitor of SMC migration and proliferation.	Acute coronary syndrome, HIV-associated nephropathy, Diabetic nephropathy	[26, 167–169]
		Podocan-like protein 1 (PODNL1)	56.5 kDa	Upregulate PI3K/Akt/mTOR signalling pathway, leading to cancer cell growth and migration in glioma.	Glioma	[27, 170, 171]

GAG- Glycosaminoglycan, RTK- Receptor Tyrosine Kinase, TLR- Toll-like Receptors. DBC- Depolarizing bipolar cells, SMC- Smooth muscle cells

2.1. Structure of Class I SLRP

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].

2.2. Structure of ASPN in Mouse and Humans

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).

Figure 1. Structural representation of class I SLRPs: decorin (DCN), biglycan (BGN) and asporin (ASPN).

All three members (DCN, BGN, and ASPN) display similarities in structure with 14 amino acid signal peptide (SP), 16 amino acid pro-peptide (PP) (ASPN has 18 amino acid propeptide), 4 N-terminal cysteine residues with one chondroitin or dermatan GAG sidechains in DCN, two in BGN and none in ASPN, 10-LRRs with N-linked oligosaccharide and 2 C-terminal cysteine residues. Additionally, ASPN contains a unique stretch of D-repeats (typically 13) at its N-terminal. The core DCN protein acts as a tumor suppressor by inducing p21 by interacting with EGFR and also modulates TGF-β signaling. In contrast, BGN acts as a tumor promoter by inducing TLR/NF-κB and MAPK signaling. On the other hand, ASPN acts as both a tumor-promoter and tumor-suppressor, depending upon the tissues.

2.3. Structure of Class II SLRP

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].

2.4. Structure of Class III SLRP

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].

2.5. Structures of Class IV and V SLRP

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].

3. Genetic variations/mutations of ASPN in cancer and bone-related diseases

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.

4. ASPN function and its associated mechanisms in cancer and other diseases

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).

Figure 2. Signaling pathways influenced by ASPN in cancer cells.

ASPN can be CAF-secreted, cellular, and nuclear localized. The secreted ASPN from CAFs interacts with TGF-βR1 in cancer cells, thereby blocking TGF-β signaling and hence suppressing tumor migration and invasion, whereas, in its interacting with EGFR and CD44, it promotes tumor progression by regulating the transcription of EMT-related genes, upregulating Bcl-2 (anti-apoptotic) and downregulating Bad (apoptotic) proteins. On the other hand, the cellular ASPN promotes TGF-β signaling and cancer progression by interacting with Smad2/3 and promoting their translocation to the nucleus to promote the transcription of EMT-related genes. Additionally, the nuclear ASPN directly interacts with LEF1 independent of β-catenin in the nucleus to facilitate the transcription of apoptosis repressor genes such as PTGS2, IL6, and WISP1, thereby promoting the survival of cancer cells.

4.1. TGFβ is the major regulator of ASPN

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].

4.2. Regulation of EGFR family members signaling by ASPN

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].

4.3. CD44 signaling and its association with ASPN

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].

5. Role of ASPN in Cancer

Mounting evidence of research supports that ASPN is expressed in ECM and inside the cancer cell cytoplasm. However, it is not fully explored how this increased ASPN level interacts with other membrane-bound molecules such as EGFR, HER2, integrins, and other membrane-bound proteins, as well as cytoplasmic proteins and transcription factors, to elucidate its action in cancer fully. In this subsection, we briefly discuss the role of ASPN and its associated mechanism in each cancer and other diseases (Figure 3). To obtain a holistic view, we performed in silico analysis and compared the expression analysis of ASPN along with other Class I SLRPs, DCN, and BGN, among normal tissues of adipose, blood vessels, fallopian tube, heart, muscle, nerve cell, pituitary gland, small bowel, spleen and vagina, and cancerous tissues with corresponding normal tissues through MiPanda Dataset portal. ASPN mRNA expression is insignificant among normal and cancer tissues of bladder urothelial, lung, sarcoma, and thymus cancers. DCN is not significant in biliary, sarcoma, and thymus, and BGN is not significant among kidney, sarcoma, and thymus cancers. A unique observation is that there is no difference in the expression level of all Class I SLRPs among normal and cancer types of sarcomas and thymus (Figure 4).

Figure 3. ASPN and its differential signaling pathways in both male and female-related cancers.

ASPN is known to regulate various signaling pathways to either promote or suppress tumor growth. Its localization can either be extracellular, cytoplasmic, nuclear, or all. In prostate cancer, the D-repeat polymorphism in ASPN affects the TGF-β1R and BMP-2 signaling, thereby promoting tumor survival and metastasis. In breast cancer, ASPN has a dual role based on its localization. The extracellular ASPN interacts with TGF-β1R, thereby blocking its dimerization with TGF-β2R, suppressing TGF-β signaling and suppressing tumor growth. On the other hand, the cytoplasmic ASPN interacts and promotes the translocation of Smad2/3, downstream of TGF-β signaling, to the nucleus, thereby supporting TGF-β signaling and promoting tumor growth and migration. Pancreatic cancer demonstrated ASPN as a secreted form from PSCs as well as PCCs to interact with CD44 on PCCs in a paracrine and autocrine manner, respectively, to activate NF-κB/p65 via AKT/ERK, thereby promoting tumorigenesis. In thyroid cancer, ASPN interacts and activates HER2 to activate further and phosphorylate EGFR and SRC to induce MAPK/PI3K signaling in supporting tumor growth and proliferation. The extracellular ASPN interacts with CD44, EGFR, and TGF-β in gastric cancer cells. Through CD44, ASPN activates Rac1 via EGFR, which induces the ERK-CD44-MMP2 pathway and promotes TGF-β signaling, which states its opposite role to that observed in breast cancer. Moreover, the nuclear ASPN in gastric cancer interacts with LEF-1 to induce WNT/LEF-1 signaling, independent of β-catenin, to inhibit the transcription of apoptotic genes and hence support the growth of cancer cells. Lastly, ASPN interacts with EGFR and FGF-2-mediated activation of FGF-2-FGFR1 to activate Src and cortactin in CRC. This leads to the formation of invadopodia that activates MMP2, hence promoting tumor migration and invasion.

Figure 4. Pan-cancer analysis of a. Decorin (DCN), b. BGN, and c. ASPN.

The violin plot shows the expression of Class I SLRPs genes decorin, biglycan, and asporin among normal and cancer tissues. The expression data was downloaded from MiPanda, which includes GTEX and TCGA datasets. Plots were generated in “R” software and statistical analysis, and p-values were calculated using the Wilcoxon test (Significant levels: * (p <= 0.05), ** (p <= 0.01), *** (p <= 0.001), **** (p <= 0.0001)).

5.1. Role of ASPN in hormone-related cancers

5.1.1. ASPN in Prostate Cancer (PCa)

Clinically, PCa is the second most critical cancer, leading to mortality in males. Many patients manifest an aggressive disease with metastasis and progression, while others manifest a slow/indolent disease with a low propensity to advance [55]. These tumors are assessed histologically using the Gleason score, which determines how similar the biotic prostatic specimen is to the healthy prostate gland [56]. During early prostate development, ASPN was demonstrated to be induced by androgens along with coordinated expression of androgen receptor (AR), Sox9, and Foxf2 [57]. ASPN was shown to be one of the androgen-responsive genes and was also seen encircling tumor cells in the stroma of a tumor, indicating a potential role in tumor progression [57]. Using immunohistochemistry (IHC), it was discovered that some regions of the human fetal prostate mesenchyme close to the prostatic bud-forming process expressed ASPN in a localized manner [34]. ASPN was identified in the TME as a possible modulator of metastatic development [33]. Men with advanced PCa have higher amounts of ASPN in their blood [58]. It has been demonstrated that polymorphisms in the ASPN D-repeat length variably affect TGF-β1 and Bone Morphogenetic Protein 2 (BMP-2) signaling. Interesting, bone lesions and the onset of PCa have been linked to the decrease of TGFβ responsiveness in prostate stromal cells [33]. In the TME of PCa, BC, small-cell gastric, and PC, ASPN is equally expressed in reactive stromal cells, including CAFs. Due to variations in ASPN’s D-repeat domain length, its expression in the TME may have a dual function in controlling the course of cancer. The aggressiveness of nearby local cancers and poorer oncologic outcomes are linked to ASPN’s expression in prostate reactive stromal cells. A higher risk of metastatic progression was found for ASPN with 14 D-repeats (ASPN D14), but a lower risk was found for ASPN with 13 D-repeats (ASPN D13) [59]. ASPN interacts with CD44 to activate Rac1 and encourage CAF invasion. It may also transactivate Rac1 in cancer cells to increase cancer cell invasion [60]. Recently, according to the Hurley group, the single-cell RNA sequencing analysis of benign, invasive cribriform, and intraductal carcinoma of PCa revealed that ASPN and FAP-positive tumors are associated with lethal disease along with an immunosuppressive environment [61]. These data are further supported by the Tewari group findings, showing that ASPN is one of the top-differentially upregulated genes among connective tissue genes and is associated with extra-capsular extension and lymph node invasion of PCa, resulting in poor clinical outcome and survival [62]. The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and oncomine data sets (multiple platforms), Zhang et al. demonstrated that ASPN, along with PENK and MEIS2, acts as independent factors/biomarkers associated with PCa cell growth, proliferation, and immune cells [63]. Overall, these studies are the outcome of tissue IHC or transcriptomic data. However, ASPN functional and biological mechanism supporting aggressive phenotype is lacking.

5.1.2. ASPN in Breast Cancer (BC)

Breast, lung, and colorectal cancers are among the three most prevalent malignancies in females. Early BC is thought to be treatable. Both locoregional and systemic therapy have advanced significantly over the past several years with a reduction in therapeutic intensity, thereby avoiding overtreatment, but undertreatment has also been a primary emphasis [64]. Maris et al. reported that in BC, the stroma of the cancer lesions showed strong ASPN expression, but epithelial cancer cells did not express ASPN. ASPN expression is absent in normal breast tissue [45]. In the case of BC, ASPN has been described as a tumor suppressor in patients with low-grade malignancies. On the other hand, high ASPN expression is associated with considerably higher relapse-free survival (RFS), but RFS is significantly lower in individuals with grade 3 tumors [8]. Only (hormone receptor) HR⁺ cells can significantly increase fibroblast ASPN expression. Contrarily, ASPN expression in fibroblasts is significantly suppressed by interleukin-1β (IL-1β) secreted from TNBC cells. This highlights the aggressive nature of the subtype of BC cells [45]. Several pro-metastatic and pro-angiogenic proteins, including VEGFα, MMPs, TNF, and most notably TGF-β1, are expressed when IL- β1 is present [65]. The observations made by Blomme et al. suggest that ASPN prevented the phosphorylation of the Smad2 protein brought on by TGFβ-1 in tumor cells. ASPN also lowered the number of stem cells in several BC cell lines and inhibited cells from undergoing EMT. Moreover, a peptide fragment with the amino acids 159 to 205 was necessary for the inhibitory action of ASPN [66]. Therefore, when xenografted into mice, TNBC cells that express ASPN develop noticeably more slowly and with reduced invasiveness. Therefore, ASPN acts as a tumor suppressor in TNBC by blocking TGFβ-1 signaling tumor cell migration through EMT [45]. A recent report by Castellana et al. demonstrated that ASPN is one of 56 differentially upregulated genes analyzed between ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Interestingly, a cell adhesion molecule GJB2 (connexin 26) was upregulated in DCIS and IDC relative to normal breast tissues and correlated with increased ASPN levels in DCIS and IDC. This data indicated that the association of ASPN and connexins will occur in the early stage of carcinogenesis or cancer initiation to promote migration, invasion, metastasis, and cancer dissemination [67]. Overall, the contradiction between tumor promoter versus suppressor function of ASPN in BC must be investigated in mice models.

5.2. Role of ASPN in gastrointestinal (GI) cancers

5.2.1. ASPN in Gastric Cancer (GC)

Gastric cancer (GC) is among the most diagnosed malignancies worldwide, especially more prevalent in East Asia. GC is mostly diagnosed at advanced stages due to few potential diagnosis approaches. ASPN is abnormally expressed in GC, as observed by Zhang et al. [68], and so it can be a potential biomarker for diagnosing GC. In scirrhous GC, which is known for the worst prognosis among all the types of GCs, ASPN is released from CAFs, interacts with CD44, and activates Rac1, which facilitates cancer cell invasion via paracrine signaling [46]. Ding et al. demonstrated that in GC, ASPN suppression causes the downregulation of Bcl-2, the anti-apoptotic protein, while the upregulation of Bad, the pro-apoptotic protein. Also, the reduced ASPN expression suppressed the expression of migration-related proteins such as CD44 and MMP-2. Further, ASPN interaction with EGFR in GC promotes the expression of p-EGFR and p-ERK, not total EGFR and ERK. Therefore, by stimulating EGFR and the ERK-CD44/MMP-2 pathway, ASPN induces GC metastasis [32]. ASPN also regulates GC cell proliferation by interacting with proteasome 26S subunit non-ATPase 2 (PSMD2), which is required for suppressing phosphorylation of ERK, AKT, and p38 kinases [69]. Further, ASPN blocks GC cell’s apoptosis and promotes their proliferation and migration by binding to lymphoid-enhancer binding factor-1 (LEF-1), independent of β-catenin, which is a co-factor in the Wnt/LEF1 pathway to promote gene transcription for GC cells survival, growth and proliferation[68]. As mentioned earlier, the tumor suppressor role of DCN in binding with TGFβ in normal gastric cells and blocks its signaling. Also, ASPN is observed to be a tumor suppressor in TNBC by inhibiting TGFβ signaling. However, it was first observed by Basak et al. in GC that ASPN interacts with TGFβ and induces downstream Smad2 phosphorylation, thereby promoting tumor growth. ASPN was also shown to upregulate EMT signaling and metastasis in GC as the increase in mesenchymal proteins such as N-cadherin and fibronectin and reduction in epithelial markers such as E-cadherin was observed. A recent study by Liu et al. also demonstrated 12 genes as top differently expressed genes in GC compared to adjacent tissues with a correlation with overall survival. ASPN is one of the genes associated with EMT signaling. GC has a poor prognosis due to a lack of early diagnosis and prognostic markers. Hence, an integrated in silico analysis employing differentially expressed genes and a protein-protein network between GC, and normal gastric tissues was created. The study identified 9 gene signatures, including ASPN as hub genes for predicting better survival and targeting GC [72]. Another GEO database analysis of GC validated that 21 core genes associated with prognostic value and ASPN emerged as the top gene, along with FAP and CTHRC1 relevant to immune infiltration and macrophage markers expression [73]. Earlier, gene set enrichment analysis (GSE29272) also reported ASPN as a hub gene demonstrated to have a predictive value associated with overall and disease-free survival in GC patients [74]. Therefore, ASPN can be an important prognostic and diagnostic marker for GC, and inhibiting ASPN-TGFβ and PSMD2 interaction can be an effective therapeutic strategy to treat GC patients.

5.2.2. Role of ASPN in Colorectal Cancer (CRC)

CRC is the fourth most prevalent cause of cancer mortality, accounting for 9.2% of deaths globally. It is the second most common adult cancer in women and the third most common in men [75]. The extracellular or secretory ASPN was abundantly expressed in CRC tissues compared to the normal tissues and is strongly correlated with the patients’ lymph node metastasis status and TNM (tissue, node, metastasis) stage. Wu et al. discovered that knocking down ASPN in CRC cell lines RKO and SW620 (high ASPN-expressing cells) or overexpressing in cell lines HT-29 and LoVo (low ASPN-expressing cells) increases the migration, invasion, and metastatic characteristics of the CRC cells. Also, ASPN stimulated the VEGF (vascular endothelial growth factor) signaling pathway in human umbilical vein endothelial cells (HUVECs), which facilitated the development of the tumor, and its overexpression in HT29 cells induced liver metastasis. The knockdown of ASPN in RKO cells reduced the incidence of liver metastasis. As explained before, ASPN stimulates the expression of pEGFR at Tyr1173, its downstream product pSRC at Tyr416, and pCortactin at Tyr421 [76]. Src plays a significant role in tumor progression and metastasis in various cancers and becomes hyper-expressed and -activated following certain stimulation or dysregulation of certain growth receptors such as EGFR, HER2, PDGFR, FGFR, and VEGFR [77]. Awata et al. showed that ASPN directly interacts with FGF-2 and facilitates the formation of the FGF-2-FGFR1 complex [78], which induces the activation of Src. Further, it was demonstrated by Zhang et al. that TGF-β1 inhibits the phosphorylation of cortactin [79], and ASPN is known to inhibit TGF-β1 by directly interacting with it. Also, EGF and PP2 blocked the cortactin phosphorylation, indicating that activation of cortactin is due to ASPN-mediated EGFR/Src phosphorylation [76]. The activated cortactin leads to invadopodia formation and increased secretion of MMPs [80]. Hence, ASPN enhances the EGFR-Scr-cortactin pathway to induce tumor proliferation and metastasis in CRC.

Apart from the role of ASPN as an ECM constituent secreted by CAFs, its role as an intercellular protein also has great significance in promoting cancer. Li et al. demonstrated that the EGFR pathway alone could not drive the CRC progression, and therefore, changes in the TGF-β pathway were observed. It has been previously described that secretory ASPN inhibits the TGF-β pathway by blocking the TGF-β1 receptor. However, Li et al. observed the TGF-β pathway promoting the role of ASPN in its intercellular form in CRC. The cytoplasmic ASPN promotes p-Smad2/3 and further N-cadherin and downregulates E-cadherin expression. Also, ASPN, though its LRR domains, interacts and facilitates the translocation of p-Smad2/3 to the nucleus by recruiting them on the nuclear membrane, thereby enhancing the mRNA expression of EMT-related molecules such as twist, FOXC2, and zinc finger E-box binding homeobox 1 (ZEB1) [44, 81]. Hence, the extracellular ASPN has a completely different role of inhibition of the TGF-β pathway than the cellular or cytoplasmic ASPN, which promotes TGF-β signaling by promoting the transportation of p-Smad2/3 to the nucleus and transcription of EMT-related genes, promoting CRC cells invasion. Also, the extracellular ASPN promotes the EGFR/Src/cortactin pathway to induce metastasis and migration in CRC.

5.2.3. Role of ASPN in Pancreatic Cancer (PC)

The third most common cause of cancer-related deaths in the United States is pancreatic ductal adenocarcinoma (PDAC). This cancer has a poor prognosis because of its rapid local development and early systemic dissemination. Most patients show metastatic or incurable illness upon diagnosis [82]. An important characteristic of PC is the development of excessive desmoplasia stimulated by pancreatic stellate cells (PSCs). PSCs, a type of CAF cells in TME of PC that interact with cancer cells as well as other stromal cell types, including immune, endothelial, and neuronal cells, to create a microenvironment that is favorable for pancreatic tumor growth and allows for both local tumor growth and distant metastasis [83]. These PSCs usually remain quiescent under a healthy state but get activated in a diseased state on stimulation by factors such as cytokines, non-coding RNAs, oxidative stress, hyperglycemia, ion channels, and calcium signaling [84]. This changes their phenotype to myofibroblasts [85] and secrete an excess of ECM components, including ASPN, which shows high expression in PC ECM [86]. ASPN is a potential ligand for activating the CD44/Rac pathway, as demonstrated by Satoyoshi et al. in scirrhous GC [46]. Wang et al. observed that ASPN is expressed in the cytoplasm while CD44 is on the surface of PSCs and pancreatic cancer cells (PCCs), such as MIA PaCa-2, PANC-1, and PSC cell lines. The exogenous ASPN, secreted from PSCs as well as endogenous ASPN from PCCs, interact with CD44 on PCCs in a paracrine and autocrine manner, respectively, to induce AKT/ERK signaling to further activate NF-κB/p65 [54]. The NF-κB/p65 is a transcription factor that remains inactive in the cytoplasm when bound to IKβα in a normal condition. In a diseased state, via AKT/ERK signaling, IKβα gets phosphorylated and undergoes ubiquitination-mediated proteasomal degradation [87], resulting in the release of NF-κB/p65. The activated NF-κB/p65 translocate to the nucleus to modify the transcription of EMT-related genes, such as suppressing E-cadherin and promoting N-cadherin expression [54, 88]. All these pathways promote EMT and hence promote PCC invasion and migration.

5.3. Role of ASPN in other cancers

5.3.1. Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most frequent endocrine cancer. It accounts for around 85% of all thyroid tumors with well-differentiated follicular origin. It is regarded as a slow-growing tumor with a 10-year survival rate of about 93% [89]. Lymph node metastases (LNMs) and distant metastases considerably enhance the morbidity and mortality of PTC patients [90]. ASPN has been observed to play a significant role in the invasion and migration of PTC. The study by Zhan et al. demonstrated that ASPN is co-localized with HER2 at the cell membrane and cytoplasm in PTC tissues. Therefore, ASPN interacts with HER2, leading to its phosphorylation (p-HER2). HER2 expression promotes the EGFR expression, and both are overexpressed in PTC and are associated with LNM [50, 91]. Further, p-HER2^Y1248 promotes p-EGFR^Y1171, which activates Src kinase and MAPK /PI3K signalings [92]. MAPK is the main driving factor in the progression of PTC, and the typical members of the MAPK signaling cascade include BRAF^V600E and mutated RAS [93]. Therefore, with the activation of the MAPK pathway, EMT transcription-promoting TFs such as SLUG, ZEB1, and ZEB2 are increased while the expression of E-cadherin is suppressed. Also, Src is a critical second messenger in HER2-mediated tumor invasion. The HER2-Src interaction upregulates HER2 expression as well as HER2/3 dimerization. The resulting increased expression of p-Src^Y418 activates EGFR by increased expression of p-EGFR^Y845 and p-EGFR^Y1101 [50]. Therefore, ASPN drives the activation of EMT-related TFs and genes via the MAPK pathway induced by the HER2/SRC/EGFR axis, stimulating EMT and promoting the invasion and migration of cancer cells in PTC.

6. Role of ASPN in specialized cells of ECM

6.1. Cross-talk between ASPN and Cancer Associated Fibroblasts (CAFs)

Fibroblasts are metabolically active and distinct forms of mesenchymal cells that play a key role in ECM regulation during tissue homeostasis and disease progression. Upon abnormal activation, these fibroblasts facilitate fibrotic diseases [94, 95]. In the case of cancer, CAFs play a significant role in tumor proliferation and migration. The cancer cells release cytokines such as TGF-β, growth factors such as fibroblast growth factor-2 (FGF-2), and platelet-derived growth factors (PDGFs) that aid in forming CAFs from normal fibroblasts in the tumorous tissue [96]. Recently, Itoh et al. demonstrated that ASPN is a major factor facilitating the expansion of pro-tumorigenic fibroblast from normal fibroblast cells. Interestingly, normal fibroblasts (NFs) are educated by CAFs to develop CAF-educated fibroblasts (CEFs) that generate reactive oxygen species (ROS), which further stimulates NF-κB-dependent production of inflammatory cytokines and ASPN. On the other hand, α-smooth muscle activation (α-SMA), the CAF marker, was not observed to be increased. Furthermore, ASPN upregulates the Kynurenine (KYN) pathway by inducing its downstream molecules, which causes cytocidal effects against CD8⁺ T cells, thereby causing immunosuppression. Apart from targeting CD8⁺ T cells, ASPN also upregulates CD44-Rac1 signaling to increase cancer dissemination and insulin growth factor-I (IGF-I) signaling to promote the transcription of EMT-upregulating genes and repress the transcription of EMT-downregulating genes [97] (Figure 5). Rochette et al. demonstrated ASPN as a stromal-expressed biomarker and correlated it with tumor progression. The lethal PCa subtype expresses ASPN and distinguishes the lethal subtype from the indolent subtype [60]. In PCa, reactive stroma develops during the early stage and progresses with cancer progression. It significantly regulates PCa invasion and migration [98]. The same group also observed a significant correlation between the levels of reactive stroma and ASPN. They evaluated for an increase in ASPN levels at high reactive stroma in most of their samples. Also, they observed the elevation of ASPN levels in normal immortalized prostate fibroblasts when incubated with the conditioned media from PC-3 cells and not from LNCaP cells, suggesting that the expression of ASPN in fibroblast cells is specific in lethal tumor subtype [60].

Figure 5. CEF-mediated ASPN stimulation induces CD44/Rac1, IGF-I/IGF-IRβ signalings, and CD8⁺T cell death/immunosuppression.

CAFs educate NFs to form CEFs by increasing ROS levels, which further induce HIF-1α and COX2 through NF-κB regulating and blocking the ubiquitin-mediated degradation of HIF-1α. The NF-κB signaling upregulates ASPN, CXCL6 inflammatory cytokine, IL-6 and IL-1β. ASPN promotes the expression of CD44, which leads to the CD44/Rac1 signaling pathway induced by IL-1β, leading to cancer cell migration. Apart from CD44, ASPN also stimulates the expression of KYN pathway enzymes such as IDO-1 and KYNU, resulting in increased 3-HAA, which ultimately causes the apoptosis-mediated death of CD8⁺T cells. ASPN also upregulates PAPP-A, which activates IGF-I by cleaving the IGF-I/IGFBP-4 complex and promoting IGF-I/IGF-IRβ signaling to promote the transcription of EMT-upregulating genes such as ZEB1/2 and twist leading to repression in E-cadherin (epithelial marker) and increase in vimentin (mesenchymal marker) while suppressing the EMT-regulating gene i.e. GSK-3β.

6.2. ASPN and Stem cells

Stem cells are defined as unspecialized cells in a living body that possess the ability to differentiate into various specialized cells as well as to self-renew. Based on the ability of stem cells to differentiate into single or multiple specialized cells, they are classified into totipotent, pluripotent, multipotent, oligopotent, and unipotent stem cells [99]. In TME, tumor metastasis is not just carried out by cancer cells alone; it also involves an interplay of different and multiple cell types that include CAFs, immune cells, and cancer stem cells (CSCs). In 2019, Hughes et al. demonstrated the role of ASPN in regulating the self-renewal and differentiating ability of mesenchymal stem cells (MSCs) to promote tumor metastasis [59]. MSCs are also known as multipotent stromal cells (a multipotent stem cell) that are found in several regions such as the dental pulp, bone marrow stroma, which is close to hematopoietic stem cells (HSCs), and fat [100, 101]. MSCs can differentiate into connective tissue lineages such as osteocytes, chondrocytes, and adipocytes [102]. They migrate to inflammatory sites and into the TME, interacting with cancer cells via paracrine signaling. This interaction between MSCs and cancer cells promotes EMT, leading to metastasis [103]. According to Hughes et al. in TME, MSCs possess high ASPN levels, which remain bound to BMP-4, a key player in MSC differentiation. Due to this, BMP-4-mediated Smad1/5/9 phosphorylation and subsequent signaling are blocked, which enhances MSC’s self-renewal ability while blocking its lineage differentiation. Further, they observed that in a prostate tumor of ASPN-null mice, there were very few MSCs with a significant decrease in cancer stem cells (CSCs) and high levels of CD8⁺ T cells, thereby markedly reducing metastasis compared to wild-type mice [59]. Therefore, ASPN is an important factor in driving MSCs-mediated cancer cell metastasis. In the case of a subtype of BC, it is known as a tumor suppressor gene in TNBC by inhibiting TGFβ1-mediated Smad2 phosphorylation and further EMT and stemness in BC cells [45].

6.3. ASPN and immune cells

There is a significant role of ASPN in CEF-mediated immunosuppression by repressing CD8⁺ T cells via the kynurenine pathway and tumor progression via IGF-I signaling in GC cells, as observed by G. Itoh et al. When NFs are incubated with the conditioned medium from CAFs, NFs are transformed into CEFs, leading to ROS generation and changes in hypoxic conditions that cause transcriptional reprogramming [104, 105]. This oxidative signaling induces the activation of NF-κB that stimulates HIF-1α and COX2, which control the expression of IL-1 (IL-1β and IL-33), IL-6 (Lif and Crlf1), inflammatory cytokine, i.e., CXCL6 and ASPN in a coordinate manner. Furthermore, ASPN facilitates the upregulation of indolamine 2,3-dioxygenase 1 (IDO-1), kynureninase (KYNU), and pregnancy-associated plasma protein-A (PAPP-A), thereby promoting kynurenine (KYN) pathway and IGF-I activation in CEFs respectively. Also, the upstream enzyme in tryptophan metabolism, kynurenine 3-monooxygenase (KMO), was also highly expressed. Further, the metabolic products of these enzymes, such as KYN and 3-hydroxyanthranlic acid (3-HAA), cause cytocidal effect-mediated CD8⁺T cell death, causing immunosuppression in TME [97]. ASPN also stimulates PAPP-A expression, activating IGF-I/IGF-IRβ signaling [106]. In normal conditions, the majority of IGF-I remains inactive by being bound to IGF-binding proteins (IGFBP4), thereby regulating IGF-I signaling [107]. Upon high PAPP-A expression, it cleaves the IGF-I-IGFBP4 complex, releasing IGF-I, which further interacts with IGF-IRβ and stimulates IGF-I/ IGF-IRβ signaling. The IGF-I/IGF-IRβ induces Ras/Raf/MEK/ERK to facilitate the active transcription of EMT-promoting genes such as ZEB1/2 and Twist and also activates PI3K/AKT pathway to inhibit GSK-3β, a significant EMT-downregulator. This results in the suppression of E-cadherin expression (epithelial marker) and an increase in vimentin expression (mesenchymal marker), hence causing invasion and migration of cancer cells [97, 108]. By mixing NFs and CAFs directly in mice xenografts, CEFs were produced without cancer cells. Once CEFs were produced, they sequentially educated NFs, resulting in the continued production of CEFs. ASPN^high/IDO1^high/KYNU^high/α-SMA⁻ CEFs were found in the distal invading front of diffuse-type gastric malignancies. These CEFs increased in size in the fibrotic stroma and led to the spread of cancer cells [97].

7. Possible role of ASPN in bone metastasis

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.

8. Role of ASPN in other diseases.

8.1. Role of ASPN in Osteoarthritis (OA)

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].

8.2. Possible role of ASPN in Polycystic ovary syndrome (PCOS)

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].

9. Mouse models deciphering the role of ASPN.

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.

10. Conclusion and future prospectus

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.

Highlights.

• ASPN is abnormally expressed in several cancers.

• ASPN has a dual role as a tumor promoter and suppressor in cancer.

• ASPN has several variants which are associated with cancer metastasis and osteoarthritis.

• ASPN regulates TGFβ, CD44, EGFR, HER2, BMP2, and BMP4 to promote epithelial to mesenchymal transition to favor cancer metastasis.

• ASPN plays a major role in CAF-epithelial cancer cell and CAF-immune cell cross-talks and mesenchymal stem cell differentiation.

• ASPN’s targeting strategies are underdeveloped.

Abbreviations

ASPN

Asporin

DCN

Decorin

BGN

Biglycan

LRRs

Leucine-Rich Repeats

TGFβ

Transforming Growth factor-β

EGFR

Epidermal growth factor Receptor

MMPs

Matrix metalloproteinases

BMP

Bone Morphogenetic Protein

PSMD2

Proteasome 26S Subunit Non-ATPase 2

FGF

Fibroblast Growth Factor

GAG

Glycosaminoglycan

KYN

Kynurenine

KMO

Kynurenine 3-Monooxygenase

miRNA

Micro RNA

CAM

Cell Adhesion Molecules

EMT

Epithelial-mesenchymal transition

TME

Tumor microenvironment

ECM

Extracellular Matrix

MSC

Mesenchymal stem cells

CSC

Cancer stem cells

CAFs

Cancer-Associated Fibroblasts

CEF

CAF-educated fibroblast

PCa

Prostate cancer

BC

Breast cancer

TNBC

Triple-negative breast cancer

GC

Gastric Cancer

CRC

Colorectal cancer

PDAC

Pancreatic ductal adenocarcinoma

GBM

Glioblastoma

OA

Osteoarthritis

PCOS

polycystic ovary syndrome

LDD

Lumbar-Disc Degeneration

DCIS

Ductal Carcinoma In Situ

IDC

Invasive Ductal Carcinoma

RP

Radical Prostatectomy

TCGA

The Cancer Genome Atlas

GEO

Gene Expression Omnibus