The aspects of CCN protein family on breast cancer

: CCNs are specific type of matricellular proteins, which are essential signaling molecules, and play multiple roles in multicellular eukaryotes. This family of proteins consists of six separate members in mammals. The architecture of CCN proteins is multimodular and comprises four distinct motifs. CCN proteins achieve their specific physiological functions by binding to integrin receptors. The CCN family has been implicated in both cure and disease with impacts on biological interactions, such as cell adhesion, chemotaxis and migration, mitogenesis, cell survival, angiogenesis, differentiation, tumorigenesis, immune functions, chondrogenesis, and wound healing. Breast cancer is the most commonly diagnosed cancer worldwide and the leading cause of cancer mortality among women triggered by atypical expression of CCNs. A favorable or unfavorable association between various CCNs has been reported in patients with breast carcinomas. Aberrant expression of CCN1 intensifies the proliferation of epithelial cells that line the lobes and ducts of the breast. Evidence also shows that the expression of CCN2 can ameliorate tumor growth and metastasis. However, CCN3 (NOV), CCN5 (WISP-2), and CCN6 (WISP-3) are consistent with neoplastic development and metastasis repression. Particular CCN members can develop tumors and cancer progression, whereas others can competitively counter the processes. Several studies have been conducted on CCN proteins and cancer in recent years. In our study, we intend to provide an overview of those research works while keeping breast carcinoma on focus. We believe that the importance of the CCN protein family in breast cancer should be reconsidered.


INTRODUCTION
Members the CCN family are matricellular proteins that are present in the extracellular matrix (ECM). Unlike most other ECM proteins, they serve a regulatory role instead of structural [1]. The CCN family comprises six homologous proteins (CCN1-CCN6).
They share a conserved motif and mosaic structure, yet serve diverse functions, with CCN5 as the only exception [2]. The first three (CCN1-CCN3) of CCN proteins provided the acronym CCN [3]. The other members included WISP-1 to WISP-3 ( Figure   2). Breast cancer is the most frequently reported cancer, and the leading cause of cancerrelated mortality in women globally [4]. The human breast is composed of lobes that produce milk and ducts that carry the milk to the nipple, and all of these are surrounded by muscle and fat tissues [5]. Ductal carcinoma and lobular carcinoma are the predominant types of breast cancer. However, carcinomas of the connective tissues surrounding the lobe and ducts are not defined as breast cancer, as they are not unique to the breast. Furthermore, invasive ductal carcinoma is more aggressive than invasive lobular carcinoma [6][7] [8].
Since CCN proteins have diverse functionality, their involvement in cancer and breast cancer, in particular, lacks uniformity. CCN1 and CCN2 are closely associated with cell proliferation and migration, and thus, can contribute directly to tumorigenesis and tumor development [9] [10]. Increased expression of CCN1 and CCN4 is associated with breast cancer metastasis through the lymphatic vessels that surround the lymph nodes connecting the breast in the armpit [11] [12]. CCN3, CCN5, and CCN6 play an antiproliferative role, where their expression can inhibit breast cancer growth, and their absence can induce carcinoma [13] [14][15] [16]. In breast carcinoma, the positive or negative relationship of CCNs (CCN1-CCN6) has been specified in this review. The association of CCN proteins with numerous activities indicates that any alterations or malfunction of CCN protein activity can trigger a cascade of events in human cells. This information would help in designing drugs and therapeutics targeting CCN proteins in breast cancer.

NOMENCLATURE
The six matricellular proteins (CCN1-CCN6) belong to the CCN protein family ( Figure   1). Several genes encoding the CCNs are documented to be composed of four motives (cysteine-rich) with a well multimodular structure (conserved). It is estimated that the N-terminal motif bears the consensus sequence (GCGCCXXC), which is highly conserved and has IGF-binding ability. Additionally, this sequence is followed by an Nterminal signal peptide [17] [18][19] [20]. The multimodular organization implies that CCNs can act reciprocally with other proteins to exercise similar functions. CCNs' biological functions may be dependent on the availability of interacting substrates and proteins. This would also describe the peculiar biochemical consequences and the wide spectrum of pathological and physiological roles of the members of the CCN family.
The CCN protein family is known to mediate epithelial-stromal cross-talk. Therefore, they interact with some key signaling molecules, especially Notch1 and cell surface integrins [19] [21]. The position of CCN proteins is well documented in the ECM, although a variety of experiments have shown that some of them may function intracellularly or have uncertain properties [22].

CCN GENE FAMILY
The six different members of the CCN gene family include Cyr61 (CCN1), CTGF (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6) [11] [19]. Several biological functions, such as cell growth, ECM regulation, cell adhesion, migration, wound healing and angiogenesis, and diseases including fibrosis, cancer and vascular diseases are controlled by expression of the CCN gene family [23] [24]. CCN proteins are not growth factors; but, they can modify the signaling of other molecules associated with the ECM [25]. Cyr61 (CCN1) is an angiogenic ECMassociated inducer that functions as an integrin receptor ligand, and its expression is closely related to incidence of breast cancer. CTGF (CCN2, connective tissue growth factor) was initially recognized as a protein secreted by human endothelial cultured cells.
The protein encoded by the CCN2/CTGF gene is a mitogen associated with plateletderived growth factor. NOV expression was found to be an integrative site for various types of tumors. CCN4 (WNT1) is a matricellular protein with a cysteine-rich motif.

5
The WNT1 gene is involved in tumor development through the activation of β-catenin, and is considered to be a novel target against toxic cell degeneration. WNT1 is activated in the Wnt/β-catenin signaling pathway, mostly as a controller that is correlated with cellular function [26] [27].
CCN5, previously known as WISP-2, rCop-1, COP-1, HICP and CTGF-L, can perform a regulatory role by reducing epithelial-to-mesenchymal transition (EMT) in both breast and pancreatic cancer cells. In noninvasive breast cancer cell lines, several genetic exposures disrupt CCN5, which is responsible for invasive cell phenotypes [14]. It was detected as a heparin-induced gene, and acts as a growth-arresting gene in vascular smooth muscle cells (SMCs) [28]. CCN6/WISP3 is rich (IGF) in the binding protein domain. Additionally, the WISP3 gene plays an important role in mitochondrial electron transport system [29].

MOLECULAR MAKEUP OF CCN
The molecular makeup of CCNs is conserved among members and various species.
CCNs have four exclusive modules with noteworthy structural characteristics and minor exceptions [30]. The IGF-IGFBP (insulin-like growth factor binding protein-like), von Willebrand factor type C (VWC), thrombospondin 1 (TSP1), and cysteine-knotcontaining (CT) are named as Modules I, II, III, and IV, respectively ( Figure 2). These modules are accompanied by a peptide of an N-terminal that is correlated with signal secretion. The characteristics of the CCNs are highly dependent on the order of the Modules. These Modules contain hydrophobic, polar, and cysteine residues [31]. CCN5 is the only exception to the alignment of these Modules in the CCN family, as it lacks the fourth CT Module. The CCNs' cysteine ratio is comparatively larger, about 10% by mass, containing 38 cysteines separated into 17 retained zones, and extended across four fields. Some dissimilarities have been observed between CCN5 (WISP-2) and CCN6 (WISP-3). CCN5 has no CT domain as it lacks 10 cysteine amino acids [32]. The two N-terminal Modules (I and II) and two C-terminal Modules (III and IV) of the CCN proteins are separated by a linker with a variable amino acid composition [33]. Modules II and III are connected by the "hinge region," which is responsive to cleavage by proteinases [21]. A conserved exon encodes these structural modules, and the CCN genes are speculated to be products of exon shuffling [34] . attach to -v -3 integrin proteins [40]. The integrins can bind with − − 3, − 5 − 1, − 6 − 1 [41]; but, it is unclear whether CCN family members exhibit preferential binding to alpha or beta integrin subunits [42]. CCN1 and CCN2 are ligands of integrins alpha-II beta-3 in the platelets and alpha-M beta-3 in monocytes [38].
CCN1 and CCN2 also bind to heparan sulfate-containing proteoglycans (HSPGs) through a heparin-binding domain [43]. CCN2 interaction with tyrosine kinase is also reported [44]. CCN3 may function by direct binding to integrin receptors and other receptors such as NOTCH1 and fibulin-1C (FBLN1). Deregulated healing of lesions is also a risk factor in cancer progression. In the early stages of neoplastic transformation, inflammation is responsible for speeding up the development of primary neoplasia into cancer by inducing mutagens, such as reactive oxygen species [36]. Aberrant expression of CCN proteins is responsible for the regulation of diverse inflammatory mediators, including TGF-β, prostaglandins, and ECM enzymes. Moreover, estrogenic influence, pro-inflammatory adipokines such as tumor necrosis factor-alpha (TNFα), and leptin are also reported to be tumor-associated biochemical risk factors [53]. In breast tumors, both the steroid-dependent proteins-CCN1 and CCN2-are overexpressed; these proteins are also estrogen-inducible [54].
Estrogenic GPR30 signaling promotes the proliferation and migration in breast cancer cells via CCN2 [55]. CCN family members play a crucial role as downstream mediators of estrogen-and progesterone-regulated cell proliferation [54]. A recent study has shown that CCN1, CCN2, CCN3, and CCN4 are pro-tumorigenic and may be responsible for human breast carcinoma, while CCN5 and CCN6 have anti-tumorigenic effects ( Figure 2). Additionally, abnormal expression of CCN proteins increases proliferative activity and induces tumor formation [11] [56].

CCN1: CYR61
Aberrant Cyr61 expression has been observed in various classes and categories of cancer [57]. from CCN1 in MCF7 cells were induced, and CCN1 was adequate to produce antiestrogen resistance and estradiol independence [56] [58]. These studies concluded that CCN1 was implicated in the progression to more advanced stages of breast cancer [11]. Therefore, CCN1 is currently known as one of the potential targets for chemotherapy against breast cancers [30].

CCN2: CTGF
Increased CCN2 expression leads to increased breast cancer metastasis [59]. Jiang  It has been shown that CTGF stimulates cell proliferation and mediates GPR30-induced cell migration. The stimulation of GPR30 by OHT also induces CTGF in fibroblasts obtained from breast tumor biopsies, which may be related to the development of invasive breast tumors in response to endogenous estrogens or administration of OHT used for endocrine therapy [55]. Moreover, an in vivo study documented that neutralization of CCN2 using CCN2-specific antibodies reduced bone metastasis [60].

CCN3: NOV
CCN3 is involved in tumor development, which was amongst the first member to be identified of the CCN family [2]. The various biological features responsible for the Nephroblastoma gene (Nov) represent the regulatory action of this growth that extends beyond the normal response to growth stimulation [19]. The antiproliferative protein CCN3 could be an oncogenic or tumor suppressor depending on the type of tumor.
CCN3 gene may be transcriptionally upregulated by the tumor suppressor p53 [53]. A marginal and non-significant reduction in expression of CTGF has been observed in TNM stage-2/3/4 tumors than in TNM stage-1 tumors. The changes in the levels of Nov transcripts were similar to those of CTGF, except that TNM stage-4 tumors showed significantly lower levels of Nov than TNM stage-1 tumors (P ¼ 0:0048) [56]. Further, any change in CCN protein structure or expression is associated with cancer development; CCN3 is associated with tumorigenesis in non-solid cancer types. that CCN proteins can act as oncogenes or tumor suppressors, depending on the type of cancer considered [62]. Additionally, CCN3 is the most studied CCN protein for tumor typing and prognosis [2].

CCN4: WISP-1
Limited literature related to CCN4 indicates that it has pro-tumorigenic features and functionality in breast cancer. In breast cancer samples, the expression of CCN4 mRNA was increased compared to that in standard breast cancer. High and low CCN4 mRNA levels in breast cancer patients were associated with poor outcomes [12] [63]. C CN4 overexpression has facilitated the development of MCF7 tumors in xenograft studies [64]. In breast carcinoma, WISP -1 levels were lower in tumor cells than in normal breast tissue, which were comparatively less than in more invasive tumor types. In contrast, in invasive breast cancer, enhanced expression of WISP-1 is related to oncogenic transformation [53]. antagonistic breast cancer cells [67]. Moreover, the CCN6 protein is considered a newer regulator of the epithelial phenotype in breast cancer [68]. CCN6 can also act as a tumor suppressor in breast carcinoma [35]. Based on the study by Kleer et al., CCN6 protein has recently been shown to play an important role in the development and progression in breast cancer [69].

CONCLUSION
The role of CCN proteins is studied in development, angiogenesis and fibrosis, but there is convincing evidence of their critical and vital role in breast tumorigenesis. The expression profiles of CCN proteins vary in the breast tissue. It is evident that numerous cellular functions are regulated by CCNs. Even though all six members of CCNs share the same protein structure, their role is strictly regulated [59]. Breast cancer often develops from several sources such as an E2 -dependent, non-metastatic, antiestrogensensitive phenotype to an E2-independent, antiestrogen-resistant, highly invasive, and metastatic phenotype [70]. Thus, a single member of the CCN family can be oncogenic or tumor-suppressive depending on the nature of the breast tumors [53].  breast cancer [53]. Moreover, their roles in angiogenesis, tumor growth and other diseases are important areas for future research, and a complete understanding of their involvement in these processes may suggest novel therapeutic strategies [9].

Authors contribution
This work was a collaboration among all the authors. ASMS, TAH, HAF, and KAA designed outlines and drafted the manuscript. ASMS, KAA, MS, AM, TAH, and SKP wrote the initial draft of the manuscript. MJU, MAR, and HAF reviewed the scientific contents described in the manuscript. All authors read and approved the final submitted version of the manuscript.