The role of collagen triple helix repeat containing 1 (CTHRC1) in cancer development and progression.

Though CTHRC1 was initially described for its vascular remodeling activity in the abnormal neointima and adventitial of rats, its elevated expression and association with tumor microenvironment (TME) have made it a target of interest in cancer research [ 3 , 6 ]. It has been found to promote tumor progression through positive regulation of tumor spread, invasion, migration, adhesion, and metastasis of many solid tumors [ 7 – 10 ]. A limited number of earlier published review articles have explored the role of CTHRC1 in cancer [ 3 , 11 , 12 ]. Since then, a significant number of new studies have described the role of CTHRC1 in different types of cancers. Also, to our knowledge, no reviews have discussed the role of CTHRC1 in TME and immune signaling. Below, we have discussed the role of CTHRC1 in various cancers as well as its associations with key cancer-driving signaling/processes. Additionally, by exploring its interactions within TME and its influence on immune signaling, we provide a more holistic perspective on the implications of CTHRC1 in the intricate network of cancer-related phenomena. CTHRC1 has consistently been shown to be overexpressed in all the cancers where it has been studied so far. A comprehensive overview of CTHRC1 expression status in various cancers, accompanied by molecular findings and affected/associated molecules is detailed in Table 1 . CTHRC1 has been found to correlate with advanced tumor-node-metastasis, increased lymph node and tumor size, and decreased overall survival (OS) and disease-free survival (DFS). A brief description of specific cancers with enhanced expression of CTHRC1 is provided below. Melanoma: Higher expression of CTHRC1 has been found in melanoma cells and associated fibroblasts, as well as in the endothelium of tumor blood vessels [ 4 ]. Tang and colleagues identified higher CTHRC1 expression in invasive primary melanomas compared to noninvasive melanoma, and even higher in metastatic melanoma compared to nonmetastatic lesions [ 13 ]. Pancreatic cancer: Liu et al. demonstrated that high CTHRC1 expression correlates with decreased survival time for pancreatic cancer patients, and vice versa. They found that CTHRC1 expression, age, tumor size, lymph node metastasis and histology were all independent predictors of pancreatic cancer after pancreatectomy [ 14 ]. CTHRC1 has also been found to be higher in hypodense groups where cancerous pancreatic tissue has a decreased blood supply [ 15 ]. Prostate cancer: Ma and colleagues demonstrated that CTHRC1 was up-regulated in prostate cancer clinical tissues and cell lines, and correlated with prostate cancer proliferation, invasion, and migration [ 16 ]. In an attempt to identify molecular pathways/biomarkers clinically relevant in metastatic prostate cancer, Bacolod and Barany found that CTHRC1 along with other secretary proteins (APLN, ANGPT2, ADAM12) were upregulated in prostate cancer, and thus suggested as a potential diagnostic marker [ 17 ]. Breast cancer: Wang et al. demonstrated that CTHRC1 is overexpressed in many different tumors including breast cancer, and its elevated expression relates to poor prognosis [ 18 ]. Li et al. found that in patients with breast cancer, CTHRC1 levels in serum and tissues were much higher than in healthy patients. They also found that cancer-associated fibroblasts (CAF) secreted higher amounts of CTHRC1 in breast cancer than normal fibroblasts [ 19 ]. While investigating the role of CTHRC1 in breast cancer, Lai and collaborators found a correlation between heightened CTHRC1 levels and aggressive clinicopathological characteristics, as well as reduced postoperative survival rates [ 20 ]. They suggested CTHRC1 as an independent prognostic factor categorizing breast cancer patients with CTHRC1 high expression into the poor prognosis group. Similarly, Kim and colleagues found an association of overexpression of CTHRC1 with poor prognosis in invasive ductal carcinoma which is the most common type of breast cancer [ 21 ]. Colon/colorectal cancer: CTHRC1’s higher expression has been noticed in colon/colorectal cancer as well as its association with poor prognosis and key cancer-supporting signaling [ 22 – 37 ]. Zhang and colleagues identified CTHRC1 as a metastasis-linked gene promoting liver metastasis in colorectal cancer. They discovered that CTHRC1 promoted hepatic metastasis through reshaping infiltrated macrophages using TGF-β signaling [ 27 ]. Hepatocellular carcinoma: Tameda et al. investigated the role of CTHRC1 in hepatocellular carcinoma (HCC) and found that CTHRC1 was overexpressed in HCC (more specifically in invasive areas) compared to normal or liver cirrhosis tissues. Moreover, CTHRC1 was much more commonly found in poorly differentiated HCC than in well-differentiated HCC [ 38 ]. Chen et al. found that CTHRC1 overexpression in HCC was associated with larger, more advanced tumors as well as metastasis. Through the induction of Integrin β1 expression and activation of focal adhesion kinase (FAK), they found that CTHRC1 promoted the adhesion of cancer cells to the extracellular matrix, suggesting that CTHRC1 enhances adhesion and migratory abilities of cancer cells, therefore promoting metastasis [ 39 ]. Overall, elevated expression of CTHRC1, accompanied by cancer-supporting signaling, has been identified in multiple cancers including bladder, cervical, endometrial, ovarian, esophageal, gastric, glioblastoma, lung, and various others, as outlined in Table 1 . These studies suggest a potential role for CTHRC1 in cancer progression and aggressiveness. Thus, CTHRC1 holds promising potential as a diagnostic biomarker and prognostic indicator. CTHRC1 has been identified in association with signaling molecules that promote various cancer-supporting functions, including cell migration, invasion, angiogenesis, and more (reviewed in [ 3 , 11 , 12 ]). The precise role and interaction mechanisms of CTHRC1 with other proteins vary depending on the specific cancer type and the contextual environment. Using Ingenuity Pathway Analysis (IPA; Qiagen), a powerful bioinformatics tool that relies on a knowledge base derived from a comprehensive collection of scientific literature and other sources, we explored how CTHRC1 interacts with other proteins in multiple cancers [ 40 ] ( Figure 1 ). Here, CTHRC1 was found to be directly/indirectly interact with a number of proteins that are predicted to be associated with cellular homeostasis, cell proliferation, cell survival, apoptosis, tumor growth, epithelial-mesenchymal transition (EMT), tumor invasion, cell migration and metastasis. It is important to note that these cancer-related processes are interconnected, however, transition between them is not always linear. The connection of CTHRC1 with key cancer-related processes is presented in Figure 1 and discussed below. CTHRC1 has been identified as a mediator in the migration and invasion of melanoma cells [ 4 , 13 ]. It has been implicated in facilitating the adhesion of cancer cells to the extracellular matrix (ECM) as well as other cells. This involvement contributes significantly to the capacity of cancer cells to invade surrounding tissues and metastasize to distant sites [ 41 ]. CTHRC1 is regulated coordinately with fibronectin and Integrin β3 by pro-invasive and -angiogenic transcription factor NFATC2 [ 4 ]. Employing metastatic melanoma cell lines MMAN (which lack endogenous CTHRC1) and MMRU (which possess high endogenous CTHRC1), and overexpression and knockdown strategies, respectively, Ip et al demonstrated that CTHRC1 enhanced the ability of melanoma cells to adhere, survive, and multiply [ 41 ]. Moreover, CTHRC1 knockdown was found to decrease melanoma cell resistance to the conventional chemotherapy drug temozolomide [ 41 ]. Park et al. found significant evidence that overexpression of CTHRC1 enhanced the adhesiveness and motility of pancreatic cancer cells, allowing pancreatic tumors to metastasize to secondary organs such as spleen, liver, colon and stomach [ 42 ]. They also found that CTHRC1 overexpression enhanced pancreatic cancer cell migration and adhesion, while CTHRC1 knockdown significantly decreased both cell motility and adhesiveness. Lastly, they demonstrated that CTHRC1 induced activation of RAC1, which is a small GTPase protein known to be involved in cell migration, adhesion and cytoskeletal dynamics. They theorized that RAC1 was required for the increased motility and adhesiveness that CTHRC1 overexpression exerted on pancreatic cancer cells [ 42 ]. CTHRC1 enables Src-FAK and ERK signaling cascades to control pancreatic cancer cell adhesiveness and metastasis [ 43 ]. CTHRC1 also represses Collagen I production into the stromal environment of pancreatic cancer, suggesting that it can affect the connective tissue between organs, and metastasis becomes more likely [ 43 ], and thus suggested as a prognostic biomarker for tumor recurrence and metastasis. CTHRC1 has also been found to promote tumor angiogenesis by recruiting bone marrow-derived cells into the tumor microenvironment [ 10 ]. CTHRC1 acts through ERK signaling cascade to stimulate the release of ANG-2 into pancreatic cancer endothelial cells, then allows TIE2-expressing monocytes (TEMs) to infiltrate tissue where CTHRC1 is overexpressed. This invasion is concerning because TEMs stimulate angiogenesis by upregulating various proangiogenic enzymes, suggesting that CTHRC1 is a valid candidate for angiogenesis-dependent conditions, including pancreatic cancer [ 10 ]. As illustrated in Figure 1 , CTHRC1 has an important role in EMT and metastasis, both of which are pivotal in the progression and invasiveness of cancer [ 3 ]. EMT represents a critical step in the metastatic cascade, wherein cancer cells undergo changes that augment their capacity to invade neighboring tissues and migrate to distant sites. Notably, the lungs, liver, brain, and bone are the most frequent sites for distant metastases. Ni et al. discovered a correlation between the clinical aggressiveness of colorectal cancer patients and elevated CTHRC1 expression. They found heightened CTHRC1 staining in the advancing front within tumor buds suggesting a potential involvement of CTHRC1 in epithelial-mesenchymal transition (EMT) and metastasis [ 30 ]. This is an important finding given that tumor budding is regarded as a morphological manifestation of EMT. In addition, this study revealed that cells overexpressing CTHRC1 underwent a transition in morphological appearance, shifting from a cuboidal-like form to a fibroblastic-like shape. This transition coincided with the downregulation of epithelial markers (E-cadherin and α-catenin) and the upregulation of mesenchymal markers (fibronectin and vimentin). They also demonstrated that CTHRC1 facilitated EMT by activating transforming growth factor-β (TGF-β) signaling pathway [ 30 ]. Elevated CTHRC1 expression has also been demonstrated to induce EMT in breast cancer [ 19 ] and epithelial ovarian cancer [ 44 ]. In breast cancer cells, CTHRC1 fosters migration, invasiveness, and EMT by activating the WNT/β-catenin signaling pathway [ 19 ]. In epithelial ovarian cancer, CTHRC1 activates SNAIL via the WNT/β-catenin signaling pathway, promoting EMT [ 44 ]. Notably, CTHRC1 knockdown has been proven to impede EMT and cellular migration in Glioblastoma [ 45 ]. The involvement of CTHRC1 in EMT and metastasis makes it a promising candidate for both prognostic assessment and therapeutic interventions in cancer. The biological impact of CTHRC1 on tumor proliferation and metastasis is associated with various cancer signaling pathways that play pivotal roles in cancer development and progression. The interplay of CTHRC1 with cancer-associated signaling molecules is depicted through IPA in Figure 2 . This illustration highlights the direct connections of CTHRC1 with the most prominent entities predicted based on IPA knowledge. The interacting signaling molecules cover a broad range of functional classes, encompassing enzymes, kinases, growth factors, transporters, transcription regulators, and more, underscoring the multifaceted nature of CTHRC1’s involvement in cancer-related processes. The connection of CTHRC1 with key cancer-associated signaling molecules is presented in Figure 3 and discussed below. TGF-β signaling involves the binding of TGF-β ligands to their receptors, leading to a series of intracellular events ultimately influencing cell type-specific gene expression and cellular responses. CTHRC1 binds directly to TGF-β receptors II and III to stabilize TGF-β receptors complex and activate TGF-β signaling [ 27 ]. TGF-β signaling is highly interconnected with key cancer signaling, such as the WNT, MAPK and PI3K pathways, allowing for complex crosstalk and fine-tuned regulation of cellular responses. CTHRC1 affects TGF-β in a concentration and time-dependent manner as well as collagen I and III deposition in cancer development [ 11 ], suggesting multifaceted nature of CTHRC1’s involvement in complex cascade of events associated with cancer development. Interestingly, TGF-β has been found to increase CTHRC1 as well as to promote EMT and metastasis via activation of SMAD2/SMAD3, which are central intracellular signaling proteins in TGF-β pathway [ 30 ]. TGF-β signaling is known for its dual roles (depending on cell contexts) as a tumor suppressor (by inhibiting cell proliferation and promoting apoptosis) or as a tumor promoter (by promoting tissue fibrosis, immune suppression, and EMT). The complex interplay between CTHRC1 and TGF-β signaling highlights the challenges in developing targeted therapies as their modulation may have varying effects depending on specific context and cancer stage. WNT signaling pathways are key players in tumor signaling. WNT/β-catenin, also called as a canonical pathway, is known for its role in cell fate determination and growth regulation. WNT/planar cell polarity (PCP) also called as a non-canonical pathway is known to regulate tissue polarity and cell movement via RHOA/JNK/NLK signaling cascades [ 46 ]. CTHRC1 is positively associated with both WNT/β-catenin and WNT/PCP pathways via a positive feedback loop of β-catenin/TCF, cellular β-catenin, DPAGT1 and the activation of RhoA, RAC1, JNK and NLK (reviewed in [ 3 , 11 ]). It interacts with FZD proteins and WNT/PCP co-receptor ROR2, forming a CTHRC1-WNT-FZD/ROR2 complex to activate WNT/PCP pathway and suppress WNT/β-catenin pathway [ 47 ]. In contrast, in some cancers e.g. in colorectal cancer, WNT/PCP signaling but not WNT/β-catenin signaling has been found to be activated by CTHRC1 [ 33 ]. In non-small cell lung cancer (NSCLC), CTHRC1 has been found to induce nuclear translocation of β-catenin. Moreover, the overexpression of CTHRC1 was observed to enhance the transcriptional activity of β-catenin/TCF, while the knockdown of CTHRC1 led to a decrease in β-catenin/TCF transcriptional activity [ 48 ]. These findings underscore the role of CTHRC1 in regulating the invasiveness of cancer cells through WNT/β-catenin pathway. In the context of the Integrin β/Src-FAK signaling pathway, CTHRC1 overexpression has been found to correlate with the activation of several key signaling molecules (MEK, ERK, Src, FAK, paxillin and RAC1) which are known to promote cancer metastasis [ 42 ]. Guo et al. demonstrated that FAK was phosphorylated and activated by Integrin β3, which is crucial for CTHRC1-mediated migratory and invasive potential of cancer cells. They also found that over-expression of CTHRC1 was strongly associated with over-expression of Integrin β3, as well as advanced clinical stages of epithelial ovarian cancer [ 49 ]. Activated ERK helps downregulate collagen deposition, stimulating tumor metastasis and increasing pro-EMT transcription factor via its interaction with MMP9. Through the ERK signaling cascade, CTHRC1 stimulates the release of ANG2 to fuel angiogenesis [ 10 ]. CTHRC1 also binds WAIF1 and activates PKC-δ and ERK to stimulate osteoblast differentiation which is indicative of this protein’s role in angiogenesis and bone metastasis. In addition to these above-mentioned pathways, CTHRC1 also interacts with other signaling molecules and pathways relevant to cancer. For example, CTHRC1 has been found to enhance colony formation, migration, and invasion of hepatocellular carcinoma cells by downregulating TP53 and stimulating MMP9, an invasion-associated factor [ 50 ]. CTHRC1 has been implicated in promoting tumor angiogenesis through the activation of the PI3K/AKT/mTOR signaling that activates HIF-1α and VEGF, key players in the process of angiogenesis. CTHRC1 is also involved in cancer cell migration and invasion through the HIF-1α/CXCR4 signaling axis (reviewed in [ 3 ]). These findings highlight the multifaceted nature of CTHRC1’s interactions with cancer-associated signaling molecules. Immunotherapy has demonstrated notable success in the treatment of diverse cancer types, employing treatments such as immune checkpoint inhibitors, lymphocyte-promoting cytokines and vaccines. However, the effectiveness of these approaches is hindered by challenges related to delivery systems, distinct toxicities, non-specific systemic effects, and the emergence of acquired or primary resistance to immunotherapies [ 51 – 54 ]. Thus, the exploration of additional therapeutic strategies is imperative. The heterogeneous nature of TME makes the extracellular matrix (ECM) a key target in immunotherapy [ 52 ]. CTHRC1, as an ECM glycoprotein, is emerging as a potential target for regulating tumor-related immune signaling. The ECM comprises an intricate network of proteins influencing tumor progression through intracellular communication and homeostasis maintenance. By modulating the composition and density of the ECM, which typically includes fibrous proteins and glycoproteins, there is a potential to enhance the efficacy of immunotherapies by mitigating the immunosuppressive nature of the TME created by the ECM [ 55 , 56 ]. Thus, CTHRC1 regulation could improve patient prognosis via modulation of the TME in various cancers. Several studies have suggested a connection of CTHRC1 with the immune response in cancers, indicating a positive association of CTHRC1 with tumor-infiltrating immune cells in cancers of colon [ 24 ], gastric [ 57 – 59 ], kidney [ 60 ], breast [ 18 ], prostate [ 61 ], stomach [ 62 ], bladder [ 56 ], ovarian [ 63 , 64 ], endometrial [ 9 ], etc. The influx of immunocytes in TME associated with elevated levels of CTHRC1 in these cancers suggests a crucial role for CTHRC1 in the regulation of immune signaling. Thus, CTHRC1 inhibition may mitigate tumor-associated macrophages (TAMs) and M2-like macrophage levels, effectively inhibiting immunocytes that contribute to tumor growth and progression. Bioinformatic analyses employing Tumor Immune Single-Cell Database (TISIDB; an integrated repository portal designed to provide information on tumor-immune interactions at the single-cell level), and Tumor IMmune Estimation Resource (TIMER; a web server for systematical analysis of immune infiltrates in cancer) revealed a positive correlation of immune-infiltrating cells (CD4+ T, CD8+ T, macrophage, neutrophil and dendritic cell infiltration) with CTHRC1 overexpression in colon cancer patients, indicating CTHRC1’s role in immune dysregulation [ 24 ]. Additionally, Zhou et al. demonstrated a positive correlation between elevated CTHRC1 levels and various immune cells in both kidney renal papillary cell carcinoma (KIRP) and kidney renal clear cell carcinoma (KIRC) [ 60 ]. These findings further emphasize the association between CTHRC1 expression and immune cell infiltration across different cancer types, pointing towards the potential significance of CTHRC1 in modulating the tumor immune microenvironment. CTHRC1’s regulation of TGF-β pathway, which phosphorylates the SMAD pathway, leads to increased infiltration of TAMs. The activated SMAD pathway upregulates the secretion of CCL15, a chemokine that chemotactically attracts M2 macrophages, highlighting CTHRC1’s role in immune response recruitment in TME [ 65 ]. Interestingly, one study demonstrated a negative correlation of CTHRC1 expression with acquired immune cells, but a positive association with innate immune cells, particularly with M2 macrophage infiltration, emphasizing CTHRC1’s role in amplifying TME [ 57 ]. Tumor immune evasion is an important factor in determining cancer prognosis. Analyses with TIMER and Gene Expression Profiling Interactive Analysis (GEPIA; an interactive web server that includes RNA expression data from TCGA and GTEx projects) have shown a significant association between CTHRC1 and immunosuppressive cells such as TAM, Tregs, and M2-macrophages, associating high levels of CTHRC1 with poor overall survival in gastric cancer patients [ 58 ]. TIMER and GEPIA investigations also correlated CTHRC1 expression levels with CSF1R and CD86 of monocytes, CD68 and IL10 of TAMs, and CD163 and MS4A4A of M2 phenotype, indicating CTHRC1’s potential recruitment role in modulation of macrophage tumor infiltration [ 18 ]. Deng et. al constructed a prognostic model for gastric cancer based on eight ferroptosis-related genes, which included CTHRC1, where authors found that immune cells have a positive association with high-risk groups for gastric cancer [ 59 ]. Head and neck squamous cell carcinoma (HNSCC) is an immunosuppressive disease with high levels of CTHRC1 documented [ 66 ]. Significant associations between CTHRC1 and macrophage infiltration have been found in both HPV-positive and negative HNSCC patients, which demonstrated a negative correlation with M1 macrophages and a positive correlation with M0 and M2 macrophages [ 66 ]. Zhou and colleagues investigated the correlation between CTHRC1 and TME in prostate cancer and found that high levels of CTHRC1 were associated with increased programmed cell death protein 1 (PD-1), and programmed cell death 1 ligand 1 (PD-L1), infiltrating B cells, CD4+ cells, macrophages, neutrophils and dendritic cells [ 61 ]. It was also found that CTHRC1 correlated with MMP9, MUC1 and SLCO2B1, which are known to be associated with functions that support tumor growth and invasion [ 61 ]. A prognostic signature utilizing CTHRC1 along with other genes in stomach adenocarcinoma demonstrated a correlation with high-risk groups and tumor immune infiltration, including M2 macrophages, and TIM-3 and PD-L2 checkpoint inhibitors [ 62 ]. Zhao et al explored CTHRC1 and five other oncogenic protein expression patterns to determine ECM scores of six different datasets in bladder cancer. They found that CTHRC1 was positively correlated with high ECM and immune scores and also yielded a higher percentage of ‘responders’ to immune checkpoint blockade therapy [ 56 ]. This provides evidence that immunotherapy could be effective in cancers with high CTHRC1 expression and high ECM scores could be a screening tool to determine if immunotherapy will be effective [ 56 ]. Similar to other cancers, studies have identified a positive correlation between CTHRC1 and the infiltration density of M2-like CD68+, CD163+ and TAMs, as well as phosphorylation of STAT6 in epithelial ovarian cancer [ 63 ]. It appears that CTHRC1 has the capability to modulate the polarization of M2 macrophages, making it a potential target for immune signaling modulation [ 63 , 64 ]. In endometrial cancer, CTHRC1 has also been demonstrated to play an oncogenic role by promoting the infiltration of M2-like TAMs through its upregulation of Fractalkine chemokine receptor (CX3CR1) expression in macrophages [ 9 ]. A graphical representation elaborating the role of CTHRC1 in TME and immune signaling associated with the biological effect of CTHRC1 in cancer is presented in Figure 4 . CTHRC1’s involvement in shaping TME and modulating immune signaling across various cancers indicates its potential as a candidate for therapeutic interventions aimed at harnessing the immune system against cancer.

The regulation of CTHRC1 expression is a complex process influenced by various factors, and it may differ in different biological settings. As discussed above, TGF-β signaling has been shown to upregulate CTHRC1 expression in certain cell types and tissues [ 30 ]; however, TGF-β can also be downregulated by CTHRC1 via hampering SMAD2/3 phosphorylation. CTHRC1 is also regulated by P4HA1, a key enzyme in collagen synthesis [ 67 ]. P4HA1 knockdown reduces melanoma tumor invasion and type IV collagen. P4HA1 inhibition reduces CTHRC1 in melanoma cells in vitro and deposition of CTHRC1 in nearby tumor blood vessels in vivo [ 67 ]. To broadly understand the regulation of CTHRC1, IPA was used to predict CTHRC1 upstream regulators. As presented in Figure 5A , CTNNB1, SRF, ESR2, GATA4, NR4A3, DBN1 and FOXA2 appeared as upstream regulators of CTHRC1. These predictions are based on the following findings. ESR2 increases the expression of Cthrc1 mRNA in ectopic endometriotic lesions from mice exhibiting surgically induced endometriosis [ 68 ]. Gene set enrichment analysis suggests that ESR2 also decreases expression of mouse NR4A3 mRNA which further regulates the expression of CTHRC1 directly as well as via modulating FOXA2 [ 68 ]. NR4A3 increases Cthrc1 mRNA in mice aorta that involves angiotensin II [ 69 ]. CTNNB1 binds TCF binding site in the CTHRC1 promoter in human oral squamous cell carcinoma [ 70 ]. In a study, CTHRC1 has been found to be regulated by drebrin (DBN1) in cardiac myofibroblasts via myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). Here, siRNA-mediated inhibition of SRF or DBN1 has been found to decrease Cthrc1 mRNA expression in cultured mouse myofibroblasts [ 71 ]. Mutant mouse Gata4 protein (substitution p.S105A with its MAPK phosphorylation site mutated) decreases expression of Cthrc1 mRNA in mouse heart [ 72 ]. These upstream molecules, though not very specific and still to be experimentally validated, can be targeted to modulate the expression of CTHRC1 and downstream effects it triggers. As there are no chemical inhibitors available for CTHRC1, IPA was further used to assess if there are any chemicals that indirectly affect CTHRC1 levels. IPA predicted that CTHRC1 is modulated indirectly via SP600125, CCG-1423, THZ1 and halofuginone ( Figure 5B ). These small molecules have been studied for their effects on various cellular processes and gene regulation. The prediction that these molecules also affect CTHRC1 is based on the following findings. SP600125, which targets JNK, decreases Cthrc1 mRNA in cultured mouse cardiac fibroblasts [ 73 ]. CCG-1423, which inhibits MRTF and associated SRF signaling, decreases Cthrc1 mRNA in cultured mouse myofibroblasts [ 71 ]. THZ1, which targets CDK7, decreases Cthrc1 mRNA in failing mouse hearts [ 74 ]. Halofuginone, which is derived from Dichroa febrifuga plant, is known to inhibit SMAD3 phosphorylation and reduce CTHRC1 levels in mouse skeletal and cardiac muscles [ 75 ]. miRNAs are small, non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation by binding to the mRNA of target genes and inhibiting their expression. Studies have shown several miRNAs that are essential players in the intricate network of CTHRC1 regulation. Utilizing IPA, a comprehensive network was constructed, revealing potential regulatory interactions between CTHRC1 and various miRNAs ( Figure 6 ). IPA predicted several miRNAs that are associated with CTHRC1 regulation. Some of them are described here. miR-509-3p has been found to negatively regulate CTHRC1, and overexpression of CTHRC1 has been found to counter the suppressive effects of miR-509-3p on melanoma cell migration, invasion and EMT [ 76 ]. CTHRC1 has also been found as a target for miR-134, whose upregulation suppresses the migratory and invasive ability of melanoma cells [ 77 ]. miR155 binds to 3’-UTR of CTHRC1 and regulates CTHRC1 expression and its function in melanoma [ 78 ]. Ma and colleagues have demonstrated the binding of miR-30e-5p to CTHRC1 mRNA using dual-luciferase reporter assay, suggesting CTHRC1 regulation by miR-30e-5p [ 16 ]. miR-155 and CTHRC1 have been found to be inversely correlated in colorectal cancer with high expression of CTHRC1 and low miR-155 in carcinoma tissue compared to para-carcinoma tissue. When miR-155 silenced CTHRC1 in vitro , CRC progression and cell migratory and invasive abilities were reduced [ 31 ]. The phenomenon of multiple miRNAs targeting a single gene underscores their crucial role in finely tuning the complex and multifaceted cancer-related processes influenced by CTHRC1.

The existing body of research consistently supports the notion that CTHRC1 plays a significant role in promoting cancer progression. By influencing the extracellular matrix through various signaling pathways, CTHRC1 has emerged as a potential biomarker across various cancers. As discussed above and corroborated via a meta-analysis, lower overall survival is correlated with CTHRC1’s overexpression in various cancers [ 79 ]. Therefore, CTHRC1 is an excellent candidate for therapeutic targeting, as its regulation could positively affect patient prognosis and well-being. However, it’s noteworthy that CTHRC1 inhibition did not show an anti-tumor effect in some studies. For example, in WM239 melanoma cells xenografted mice, the knockdown of CTHRC1 in WM239 melanoma cells increased tumor growth [ 4 ]. This observation was unexpected since CTHRC1 is recognized as a mediator of melanoma cell migration and invasion [ 4 ]. One plausible explanation for this discrepancy is that CTHRC1 may play a role in regulating the switch from proliferation to invasion, given that cancer cells generally do not engage in both processes simultaneously [ 80 ]. Duarte and colleagues made a significant observation that CTHRC1 is expressed not by cancer cells themselves but by activated stromal cells within tumor or surrounding tumor cells [ 6 ]. Stromal cells, which are non-cancerous differentiating cells in tumor microenvironment, play a crucial role in providing structural and functional support to tumor and its surrounding tissues. Tumor stroma mainly consists of basement membrane, fibroblasts, extracellular matrix, immune cells, and vasculature. Although most stroma cells possess certain tumor-suppressing abilities, they change their properties during malignancy and eventually promote tumor growth, invasion and metastasis. Utilizing a transgenic mouse model, Duarte and colleagues showed that CTHRC1 is a marker for activated stromal cells in cancers; however, stromal cell expression of CTHRC1 did not contribute to its circulating levels, instead, it remains active locally [ 6 ]. This is an important observation as stromal cells are known to create a protective barrier around tumor, making it more difficult for immune cells and certain cancer treatments to penetrate tumor. Recently, stromal cells have gained attention in cancer research as potential therapeutic targets to disrupt tumor microenvironment and enhance the efficacy of cancer treatments. The expression of CTHRC1 in activated stromal cells makes it as a potential candidate for targeted therapeutic strategies aimed at disrupting stromal cells within the tumor microenvironment. There are currently no inhibitors or approved drugs that specifically target CTHRC1. However, several studies have reported promising outcomes with genetic inhibition or antibodies that disrupt CTHRC1 activity in both in vitro and preclinical animal models of cancer. For example, a recent study has reported the development of a monoclonal antibody that can block CTHRC1 signaling and inhibit the growth of colorectal cancer cells [ 81 ]. These findings underscore the potential utility of small molecule inhibitors that can interfere with CTHRC1 expression, activity, or protein-protein interactions, or block downstream signaling pathways. Further efforts to explore these avenues could pave the way for the development of targeted therapies aimed at modulating CTHRC1 and, in turn, impacting cancer progression. CTHRC1 overexpression has been associated with non-cancerous conditions as well, such as the polarization of fibroblast-like synoviocytes (FLS) and increased migration and directness of cell movement in rheumatoid arthritis patients, its negative role in myelination, and elevated expression in benign keloid scarring [ 82 ]. Given the association of TGF-β, WNT, and bone remodeling pathways with rheumatoid arthritis severity, recent studies have indicated that elevated CTHRC1 plasma levels can serve as a prognostic indicator for cartilage and bone degenerative diseases [ 83 ]. CTHRC1 is known to stimulate osteogenesis, differentiation, and function of osteoblasts. Its decreased expression is associated with suppressed bone turnover, suggesting a potential therapeutic role in treating osteoporosis [ 84 ]. Moreover, CTHRC1 has been utilized as a biomarker since it has been found to be secreted in blood, urine and saliva [ 5 , 29 ]. It holds the potential for cancer screening to diagnose malignancy early. CTHRC1 has been characterized as a prognostic marker in renal and liver cancer. However, its value as a diagnostic and prognostic biomarker for cancer should be approached with caution, as CTHRC1 overexpression has also been observed in certain non-cancerous diseases Based on the available literature, we believe that CTHRC1 may offer hope as a promising target in conjunction with immunotherapy or other cancer therapies for improved treatment outcomes. Nevertheless, a thorough assessment and comprehensive research efforts are essential to comprehend CTHRC1 expression and its interplay with different cancer-related signaling molecules, particularly in the development of anti-cancer approaches centered around CTHRC1. Future research should also focus on finding an effective CTHRC1 inhibitor and understanding the biological relevance of different CTHRC1 isoforms and multimers.

Studies have demonstrated that CTHRC1 is consistently overexpressed in a variety of cancers, and its elevated expression correlates with unfavorable prognoses, including shorter overall survival and recurrence-free survival. Studies have also shown that CTHRC1 can stimulate angiogenesis and immune responses to promote tumor growth and metastasis. While CTHRC1 is being viewed as a potential biomarker in certain cancers like liver and breast cancer, its functionality doesn’t appear to align with the classical definition of an oncogene. Indeed an overexpression of certain genes can be a normal aspect of cellular processes and the living system’s response. For example, during the immune response, certain genes may be overexpressed to help fight infections. Based on the available evidence, it appears that CTHRC1 functions cooperatively with several cancer-driver pathways toward tumor growth ultimately leading to EMT and metastatic progression. Thus, CTHRC1 has mechanistic and therapeutic importance in cancer promotion, and targeting CTHRC1 along with key cancer driver pathways could be a potential strategy for cancer management. Elevated expression of CTHRC1 has been found in other diseases/conditions like in Keloid (a cutaneous benign tumor). Thus, targeting CTHRC1 might have therapeutic values beyond cancer as well.

Collagen triple helix repeat containing 1 (CTHRC1) is a secreted glycoprotein involved in various cellular processes such as metabolism regulation, inhibition of osteoclast differentiation, and tissue repair by affecting vascular remodeling and morphogenesis. Discovered as a novel gene in adventitial fibroblasts after arterial injury, CTHRC1 was first reported in 2005 by Pyagay and colleagues as one of the highly expressed genes in rats with significant artery damage [ 1 ]. Notably, the study observed elevated CTHRC1 expression in damaged arteries, which diminished once tissue was healed, suggesting its involvement in tissue repair. CTHRC1 gene is located at chromosome 8q22.3 and can generate a 1.2kb mRNA transcript. This unique structure enables CTHRC1 to facilitate cell migration by reducing the deposition of collagen matrix. Additionally, the same structure allows CTHRC1 to attach to cell membrane promoting actin polymerization and cell polarity [ 2 ]. CTHRC1 is located in the nucleoplasm and is also localized to the extracellular matrix of injured arteries. The molecular weight of secreted CTHRC1 (28 KDa) is larger than cellular CTHRC1 (26 KDa) due to glycosylation [ 1 ]. CTHRC1 often exists as a secreted 56 KDa dimer that is not susceptible to collagenase but can also trimerize (84 KDa) and create multimers of the trimerized version that can undergo collagenase cleavage [ 3 , 4 ]. The ability of CTHRC1 to undergo collagenase cleavage may be relevant in scenarios such as tissue remodeling and wound healing, where collagen turnover is a critical aspect of the process. Thus, understanding the structural dynamics and functional implications of CTHRC1 in different forms is essential for unraveling its roles in various physiological and pathological processes. Mutant mice lacking Cthrc1 develop normally but have significant liver lipid accumulation (macrovesicular steatosis) and elevated glycogen levels in skeletal muscle and liver [ 5 ]. This suggests that CTHRC1 plays a role in regulating lipid storage and cellular glycogen levels. Its involvement in mainly remodeling processes correlates with its expression in tissues that frequently undergo remodeling, e.g. myocardium, renal arteries, epithelium, smooth muscle, and osteocytes. Because of its expression in cells with high rates of proliferation, including tumor cells, CTHRC1 has been suggested to have relevance in a cancer. However, the exact role of CTHRC1 in cancer is not well-established. In this review, we have presented a critical discussion on CTHRC1’s role in cancer development and progression, and its association with various cancer-related processes and pathways. In addition, we have discussed if CTHRC1 could serve as a potential target for cancer management.