Collagen prolyl 4-hydroxylases modify tumor progression

Abstract

Collagen is the main component of the extracellular matrix. Hydroxylation of proline residues on collagen, catalyzed by collagen prolyl 4-hydroxylase (C-P4H), is essential for the stability of the collagen triple helix. Vertebrate C-P4H is an α2β2 tetramer with three isoenzymes differing in the catalytic α-subunits, which are encoded by P4HA1, P4HA2, and P4HA3 genes. In contrast, β-subunit is encoded by a single gene P4HB. The expressions of P4HAs and P4HB are regulated by multiple cellular factors, including cytokines, transcription factors, and microRNAs. P4HAs and P4HB are highly expressed in many tumors and participate in cancer progression. Several inhibitors of P4HAs and P4HB have been confirmed to have anti-tumor effects, suggesting that targeting C-P4H is a feasible strategy for cancer treatment. Here, we summarize recent progresses on the function and expression of regulatory mechanisms of C-P4H in cancer progression and point out the potential development of therapeutic strategies in targeting C-P4H in the future.

Introduction

The extracellular matrix (ECM) is composed of several types of macromolecules, including collagen, fibronectin, elastin, laminin, hyaluronan, and proteoglycans [1]. As the most abundant component in tumor microenvironment, ECM can modify tumor cell behavior and cancer progression. ECM deposition and stiffening can enhance cancer cell growth, survival, and focal adhesion formation. In order to invade, cancer cells must first anchor onto ECM and then destroy the ECM to move into the adjacent milieu [2].

The collagen superfamily is composed of 28 members and can be separated into fibrillar and non-fibrillar collagen [3]. Collagen is widely distributed in normal tissues and tumors. As the main component of ECM, collagen can promote tumor development. For instance, Type I collagen, the main fibrillar collagen, is highly expressed in a variety of cancers and favors cancer progression via promoting tumor cell proliferation, migration, invasion, epithelial–mesenchymal transformation (EMT), and chemotherapy resistance [4–9]. Type IV collagen, the non-fibrillar collagen required for basement membrane formation, promotes tumor cell adhesion and migration [10,11]. Type VI collagen is overexpressed in human tumors and can increase the recruitment of endothelial cells to promote angiogenesis; it also increases recruitment of macrophages to promote an inflammatory response [12–14]. In addition, collagen also covers the surface of solid tumors and blood vessels and is regarded as a passive barrier to block tumor cells’ invasion. To counter this blockage, cancer cells produce matrix metalloproteinases (MMPs) to degrade the collagen and break the barrier [15–17].

Collagen is composed of three α chains and contains one or more domains contributing to the formation of collagen triple helix. The triple-helical part of collagen consists of repetitive Glycine-X-Y motifs, where the X and Y positions can be any amino acid, usually a proline or a 4-hydroxyproline, respectively. The 4-hydroxyproline residues are essential for the stability of the collagen triple helix. Collagen prolyl 4-hydroxylase (C-P4H) is the only enzyme catalyzing the hydroxylation of proline into 4-hydroxyproline at the Y position in the Glycine-X-Y motif of the α chains [18]. Recent evidence shows that C-P4H is overexpressed in a variety of human cancers and contributes to cancer progression, indicating that C-P4H may be a potential diagnostic marker and therapeutic target for cancer patients.

C-P4Hs and Cancers

C-P4H is a member of the Fe²⁺ and α-ketoglutarate-dependent dioxygenase family. C-P4H suffers oxidative inactivation during catalysis, and the co-factor vitamin C is required to reactivate the enzyme by reducing Fe³⁺ to Fe²⁺ [1,19]. Vertebrate C-P4H is composed of two α-subunits and two β-subunits and is localized within the lumen of the endoplasmic reticulum (ER). The β-subunit, a protein disulfide isomerase (PDI), is encoded by P4HB gene located at human chromosome 17aq25.3. The three α-subunits are encoded by P4HA1, P4HA2, and P4HA3 genes located at human chromosomes 10aq22.1, 5aq31.1, and 11aq13.4, respectively [20–23]. P4HAs and P4HB are translated on the ribosomes along the ER, and then the N-terminal signals of P4HAs and P4HB are cleaved upon entering into the lumen of ER. Subsequently, different P4HAs can bind P4HB to form distinct C-P4H tetramers. The P4HAs have substrate recognition and catalytic activity, and P4HB is required for the proper configuration of C-P4H to allow enzymatic activity [24]. In the absence of P4HB, P4HAs form aggregates that do not possess catalytic activity [25]. In addition to functioning as the β-subunit of C-P4H, P4HB monomers also have PDI activity. Like other ER chaperones, P4HB can also be detected on the cell surface with functions distinct from those in ER [26].

Mounting evidence has shown that P4HAs and P4HB are highly expressed in many tumors and participate in cancer progression (Table 1). It has been reported that P4HAs and P4HB can regulate tumor development via collagen-dependent or collagen-independent way (Fig. 1). Collagen interacts with integrin and activates specific signal pathway to promote cancer cells’ EMT and proliferation. Meanwhile, increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion. As the main enzyme in collagen synthesis, P4HAs could modulate cancer cell behavior via increasing collagen production. Besides, P4HAs could regulate tumor cell glycolysis, alter hypoxia inducible factor (HIF)-1α stability, modulate demethylase activity of the ten-eleven translocation (TET) and the jumonji (JMJ), or change proline hydroxylation of Carabin to promote tumor development [27–31]. As an ER chaperone, P4HB could contribute to tumorigenesis via regulating the MAPK signaling pathway, ER stress, or chaperone Bip expression [32–36]. Thus, P4HAs or P4HB may be a potential diagnostic marker and therapeutic target for cancer patients.

Breast cancer

Intra-tumoral hypoxia is common in breast cancer and is associated with a significantly increased risk of metastasis and patient mortality [37]. Type I collagen secreted by tumor cells has been identified as a marker for poor prognosis for patients with breast cancer [38]. As the target genes of HIF-1α, P4HA1 and P4HA2 upregulation induced by HIF-1α is essential for breast cancer metastasis by enhancing collagen synthesis. Inhibition of collagen synthesis and deposition by suppressing the activity of C-P4Hs have been considered as a strategy to block breast cancer progression [38–40].

Triple-negative breast cancer (TNBC) is an aggressive histological subtype with poor prognosis for cancer patients and accounts for ∼15% of all breast cancer cases [41]. Upregulated in TNBC, P4HA1 utilizes α-ketoglutarate in the proline hydroxylation of collagen, which results in reduced levels of α-ketoglutarate and increased levels of succinate. As a result, proline hydroxylation of HIF-1α is limited, leading to abated ubiquitination and degradation to increase the stability of HIF-1α. Upregulated HIF-1α enhances cancer cell stemness and further promotes cancer progression [27]. Additionally, C-P4H can act as an epigenetic modulator of cell plasticity. TET and JMJ demethylases belong to the Fe²⁺ and α-ketoglutarate-dependent dioxygenase family. P4HA2 is upregulated in breast cancer and consumes more vitamin C, which becomes less available for TET and JMJ. Reduced demethylase activity of TET and JMJ leads to enhanced global DNA and histone methylation, resulting in cell state transition and breast cancer progression [28].

Ductal carcinoma in situ (DCIS) is a noninvasive form of breast cancer. However, a part of DCIS lesions may develop into invasive cancer [42]. When assessed immunohistochemically in malignant cells and surrounding stroma of a large DCIS cohort, overexpression of P4HA2 was found to be associated with high risk of DCIS [43]. Therefore, P4HA2 can potentially be used to predict DCIS outcome.

Hepatocellular carcinoma

Increasing evidence shows that hepatocellular carcinoma (HCC) is a principal cause of cancer-related mortality globally, especially among Asian and African populations [44]. Over 50% of HCC cases are caused by Hepatitis B virus (HBV) infection, which leads to HCC development through HBV direct and indirect mechanisms [45]. One of the indirect mechanisms may involve P4HA2, which is dramatically augmented in liver samples of HBV transgenic mice and of liver cancer patients. Upregulated P4HA2 may enhance collagen deposition leading to HCC progression [46].

In addition to P4HA2, P4HB is also upregulated in human HCC tissue and cell lines. More importantly, higher P4HB levels in tumor biopsy are correlated with poorer survival of the patients [36]. Silencing P4HB inhibits HCC tumorigenesis in vivo [36]. P4HB could promote HCC cell growth, migration, invasion, and EMT in vitro through downregulating Bip [36], a chaperone highly expressed in a variety of tumors and participating in cancer progression [47,48]. How P4HB regulates Bip expression to promote HCC progression remains to be elucidated.

Besides P4HA2 and P4HB, P4HA1 has also been implicated in HCC progression via microRNA (miRNA) modulation. Decreased miR-30e level has been detected in HCC patients. It has been shown that miR-30e downregulates P4HA1 mRNA to mitigate collagen synthesis, thus abating the proliferation of HCC cells [49].

Lung carcinoma

Lung carcinoma is the world’s leading cause of cancer death [50]. P4HA2 is upregulated in lung cancer and is correlated with poor survival rate of the patients. High expression of P4HA2 could increase collagen deposition in lung cancer, which creates a stiffer ECM and accordingly triggers cancer stem-like programming and metastatic dissemination in vivo [51].

In addition to P4HA1, P4HB is also upregulated in lung cancer [52]. Although how P4HB contributes to lung carcinoma is not clear, the observation that Yiqi Chutan Formula (YQCTF, a traditional Chinese medicine) could inhibit lung carcinoma growth via decreasing the expression of P4HB suggested that YQCTF might exert its effect through regulating P4HB [52]. As demonstrated in treating lung cancer, traditional Chinese medicine, which has been practiced for thousands of years and is currently used as an alternative treatment for some types of cancers [53], warrants further investigation.

Glioma

Glioma is the most common adult malignant tumor in the central nervous system, which is classified into different subtypes, with the glioblastoma multiforme (GBM) being the most abundant and lethal [54]. P4HA1 is upregulated in gliomas, and high expression of P4HA1 is correlated with the malignancy of gliomas [55]. A study showed that P4HA1 is correlated with proliferative index and microvessel density in glioma specimens. Silencing P4HA1 inhibits the synthesis of collagen IV and disrupts the structure of vascular basement membranes in gliomas, which in turn retards tumor growth and neovascularization in xenograft models [56].

P4HA2 is overexpressed and correlated with poor prognosis for patients with glioma, too [57]. The P4HA2‒collagen‒PI3K/AKT pathway is the potential mechanism in mediating the glioma progression. P4HA2 catalyzes collagen deposition, which then interacts with collagen receptors and activates the PI3K/AKT signaling pathway, leading to glioma cell proliferation, migration, invasion, and the EMT [57].

In addition to P4HA1/2, P4HB is also highly expressed in high-grade human glioma; aberrant expression of P4HB promotes tumor invasion, angiogenesis, and growth via activating the MAPK signaling cascade, one of the main epidermal growth factor receptor (EGFR) downstream pathways that are known to be critical for glioma tumorigenesis [35]. However, the mechanism of P4HB in regulating the MAPK signaling pathways is elusive. Besides activating MAPK cascade, P4HB is also relatively upregulated in the temozolomide (TMZ, an oral alkylating agent used to treat GBM)-resistant GBM biopsies [58]. About 50% of TMZ-treated patients do not response to TMZ [59], and inhibition of P4HB attenuates TMZ resistance in malignant glioma through the PERK arm of the ER stress response [58].

Head and neck squamous cell carcinoma

Local invasion and metastasis are the main characteristics of head and neck squamous cell carcinoma (HNSCC) [60]. P4HA1 mRNA and protein levels are significantly increased in HNSCC tissues compared with those in normal tissues. High P4HA1 expression in HNSCC tissues is significantly associated with tumor grade, lymphatic metastasis, and pathological stage. High P4HA1 expression is also associated with poor overall survival (OS) and relapse-free survival (RFS) in HNSCC patients [61–63].

In addition to P4HA1, P4HA3 is also upregulated in HNSCC and is associated with poor OS. An in vitro study showed that P4HA3 promotes HNSCC cell proliferation, migration, and invasion by inducing EMT [64]. As P4HA3 has little effect on collagen synthesis [65], whether P4HA3 promotes HNSCC progression via collagen production is unclear.

Oral cavity squamous cell carcinoma (OSCC) is the most common HNSCC, which accounts for ∼3% of all newly diagnosed cancer cases [66]. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach, Chang et al. [67] found that P4HA2 expression is higher in OSCC tumor cells. Downregulation of P4HA2 reduces migration and invasion of OSCC cells in vitro. However, upregulation of P4HA2 is not associated with poorer prognosis of the patients with regard to OS and disease-specific survival.

Gastric cancer

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Many patients have inoperable disease(s) at the time of diagnosis or have recurrent disease(s) after resection with curative intention [68]. A study showed that P4HA3 is significantly upregulated in GC compared with that in normal stomach tissue, and high P4HA3 expression is correlated with unfavorable OS, probably via enhancing GC cell motility and invasiveness [69].

Similar to P4HA3, P4HB is overexpressed in 428 GC biopsies along with HIF-1α as defined by immunohistochemical staining. Patients with a high P4HB or HIF-1α expression have a shorter disease-free survival (DFS) [70]. As a transcriptional factor, HIF-1α can promote GC invasion and metastasis by upregulating P4HB expression [34]. However, how P4HB regulates GC progression has not been clarified.

Colorectal cancer

Colorectal cancer (CRC) accounts for ∼10% of all cancers diagnosed each year and cancer-related deaths worldwide [71]. Analysis of P4HA1 expression with immunohistochemistry of 599 early-stage CRC biopsies showed that patients with high P4HA1 expression had significantly shorter OS and DFS [72]. Depletion of P4HA1 leads to the decrease of CRC cells’ growth, invasion, and tumor growth in xenograft models via inhibiting collagen synthesis. Inhibition of P4HA1 with a small molecule inhibitor reduces the malignant phenotype of CRC cells and tumor growth [73].

Pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, for which mortality closely parallels incidence [74]. P4HA1 is highly expressed in PDAC. Elevated expression of P4HA1 in PDAC cells is HIF-1α-dependent and predicts a poor clinical outcome for PDAC patients [31]. Knockdown of P4HA1 significantly suppresses the glycolytic activity of PDAC cells and abates HIF-1α stability, indicating a positive feedback loop between HIF-1α and P4HA1 in PDAC. Silencing P4HA1 significantly inhibits cell proliferation, chemoresistance, and stemness of PDAC cells [31]. The molecular mechanism may be similar to that by which P4HA1 regulates HIF-1α stability in breast cancer [27]. Besides P4HA1, upregulation of P4HA2 has been detected in tissue microarrays and immunohistochemical analysis of 140 PDAC biopsies, together with Proteinase 3 (PRTN3), a protease for a variety of matrix proteins in vitro [75]. Interestingly, expression levels of P4HA2 and PRTN3 individually are not correlated with OS or DFS, but a combination of low P4HA2 and high PRTN3 expressions is associated with shorter DFS and OS, indicating a possible involvement of collagen deposition in the restraint of the tumor. This is the first report using P4HA2 and PRTN3 as biomarkers in a large number of PDAC patients from a well-annotated clinical cohort [75].

Renal cell carcinoma

Renal cell carcinoma (RCC), one of the most common malignancies in the urinary system, constitutes 3% of malignant tumors in adult [76]. P4HA3 expression is upregulated in RCC patients, and silencing P4HA3 significantly inhibits RCC proliferation, migration, and invasion. Xenograft of RCC demonstrates that P4HA3 promotes tumor growth in vivo [77].

Kidney renal clear cell carcinoma (KIRC), which is associated with high morbidity and poor prognosis, is the most common pathological type of RCC [78]. P4HB protein level is significantly higher in KIRC tissues in comparison with that in normal tissues. High P4HB expressions are significantly correlated with poor OS of KIRC patients [32].

Cervical cancer

Cervical cancer is one of the most common gynecological malignancies [79]. P4HA2 is markedly upregulated in cervical cancer tissues in comparison with that in adjacent non-neoplastic tissues. Upregulation of P4HA2 is associated with shorter OS and RFS. P4HA2 silencing attenuates cancer cell proliferation, migration, and invasion and inhibits tumor growth in a xenografted mouse model via suppressing the EMT [80]. P4HA2 could also promote cervical cancer cell glycolysis through upregulating the expressions of phosphoglycerate kinase 1 (PGK1) and L-lactate dehydrogenase A chain (LDHA), two glycolysis-related enzymes [30]. How P4HA2 regulates the expressions of PGK1 and LDHA has not been clarified. Like P4HA1, which regulates HIF-1α stability in breast cancer, P4HA2 might increase the stability of HIF-1α via regulating the levels of α-ketoglutarate and succinate, and HIF-1α upregulates the expressions of PGK1 and LDHA.

Prostate cancer

Prostate cancer is the most common malignancy and the second most common cause of cancer death among men [81]. Collagen has been regarded as a physical barrier against prostate cancer invasion. To break this barrier, cancer cells produce MMPs to degrade collagen to promote tumor invasion. Among MMPs, MMP-1 has been identified as one of most upregulated proteins [82]. Similarly, P4HA1 is overexpressed in prostate cancer and associated with disease progression. Since P4HA1 can modulate the expression of MMP-1, it is possible that P4HA1 may exert its effect on prostate cancer cell proliferation and invasion via MMP-1 [83]. Another possibility is that P4HA1 may increase the stability of HIF-1α, which then upregulates the expression of MMP-1 [84]. Targeting P4HA1 efficiently reverses the growth and metastasis of prostate cancer cells in vivo [83].

Papillary thyroid cancer

Papillary thyroid cancer (PTC) is the most prevalent form of malignancy among all cancers of the thyroid. It is also one of the few cancers with a rapidly increasing incidence [85]. Jarzab et al. [86] analyzed oligonucleotide microarrays in 50 biopsies collected intraoperatively from 33 patients with PTC and found that P4HA2 mRNA is upregulated in PTC compared with that in normal tissue. However, the function of P4HA2 in PTC has not been fully investigated.

Mechanism of P4HAs Regulates Cancer Progression

P4HAs could regulate tumor development via collagen-dependent or collagen-independent way. Here, we describe how P4HAs regulate tumor progression via collagen-independent way.

Regulation of glycolysis by P4HA1

Altered metabolism is a hallmark of cancer, and glycolysis is one of the important factors promoting tumor development [87]. Increased glycolysis is the main source of energy supply in cancer cells that use this metabolic pathway for adenosine triphosphate generation [88]. A paper by Wei et al. [87] showed that P4HA1 can be regarded as a potential modulating factor of glycolysis as its expression is highly correlated with glycolysis score and glycolysis genes. P4HA1, as the main enzyme in collagen synthesis, catalyzes proline hydroxylation of collagen by utilizing α-ketoglutarate, which results in reduced levels of α-ketoglutarate and increased levels of succinate. As a result, proline hydroxylation of HIF-1α is limited, leading to increased stability of HIF-1α [27,31]. Upregulated HIF-1α can increase the expression of glycolysis-related enzymes, which promote the progress of glycolysis [27,87]. Meanwhile, P4HA2 could also promote glycolysis through the upregulation of PGK1 and LDHA [30]. Like P4HA1, P4HA2 may increase the stability of HIF-1α. However, the regulation of glycolysis by P4HA3 has not been reported.

Regulation of epigenetics by P4HA2

Cancer cell plasticity has been proposed as an important mechanism that, along with genetic and epigenetic alterations, promotes cancer cell diversity and contributes to intra-tumor heterogeneity [89]. Transient metabolic perturbations that modify the availability of required metabolites for epigenetic enzymes emerge as candidate determinants of cell plasticity [89,90]. Epigenetic regulation, including DNA methylation and modification of histone proteins, plays an important role in malignant transformation of many cancers [91]. TET and JMJ are two members of the Fe²⁺ and α-ketoglutarate-dependent dioxygenase family. TET enzymes catalyze DNA demethylation through 5-methylcytosine oxidation [92], whereas JMJ proteins are histone demethylases [93]. It is posited that when P4HA2 is upregulated, it consumes more vitamin C. Accordingly, less vitamin C is available for TET and JMJ demethylases, leading to reduced demethylase activity of TET and JMJ. As a consequence, global DNA and histone methylation is enhanced, which results in cell state transition and breast cancer progression [28].

Regulation of proline hydroxylation by P4HAs

Proline hydroxylation, a common posttranslational modification, modulates protein folding and stability in mammalian cells [1]. In addition to collagen, P4HAs can catalyze the formation of 4-hydroxyproline on other proteins, which might regulate cancer progression. It has been reported that Argonaute 2 can be hydroxylated at proline 700 by P4HA1, and the hydroxylation increases Argonaute 2 physiological stability [94]. Argonaute 2 is required for RNA-mediated gene silencing and could regulate tumorigenesis through miRNA-dependent or miRNA-independent pathways [95], suggesting that P4HA1 might modulate cancer progression independent of collagen synthesis. In addition, Carabin can also be hydroxylated at proline 306 by P4HA2 in B-cell lymphoma cells. Carabin’s hydroxylation leads to its proteasomal degradation, thereby activating the Ras/extracellular signal-regulated kinase pathway and increasing B-cell lymphoma proliferation [29]. The abundance of hydroxyproline among the residues in animal proteins is ∼4% [27]. Thus, P4HAs might regulate cancer development via hydroxylating proline on proteins other than collagen in tumor cell.

Regulation of P4HAs and P4HB Expressions

As indicated above, the expressions of P4HAs and P4HB are frequently upregulated in a variety of cancers and are associated with a poor prognosis. It is thus important to understand the regulatory mechanisms of P4HAs and P4HB expressions to develop strategies for cancer therapies. A number of transcriptional factors, cytokines, and miRNAs have been shown to regulate the expressions of P4HAs and P4HB (Table 2).

miRNAs, the 19∼25-nucleotide short RNA molecules, play important roles in posttranscriptional regulation. In animal cells, miRNAs regulate their targets by translational inhibition or mRNA destabilization [96]. The expression of P4HA1 can be regulated by miR-30e, miR-122, miR-124-3p, and miR-124, which can regulate the progress of fibrosis, atherosclerosis, and cancer progression via silencing P4HA1 mRNA levels [49,83,97–100]. Similarly, miR-30e and Let-7g can impact HCC progression via downregulating P4HA2 mRNA levels and blocking collagen synthesis [46,101]. P4HB is a target gene of miR-210 in glioblastoma, which could modulate chemoresistance of glioma via downregulating P4HB mRNA levels [102].

HIF-1α can promote tumor growth, angiogenesis, and metastasis. P4HA1, P4HA2, and P4HB are targets of HIF-1α. HIF-1α can upregulate the expressions of P4HA1, P4HA2, and P4HB under hypoxic conditions to influence the progress of breast and GC cells [34,103].

It has been reported that p53 trans-activates P4HA2, resulting in the extracellular release of antiangiogenic fragments of collagen, which in turn inhibit tumor growth [104].

Tumor necrosis factor (TNF)-α, which is secreted primarily by macrophages, natural killer cells, and lymphocytes, also participates in ECM metabolism [105]. Zhang et al. [106] reported that TNF-α suppresses P4HA1 expression at the transcriptional level. They found that a TNF-α response element (TaRE) locates at the promoter region of P4HA1. Non-POU domain-containing octamer-binding protein (NonO), a transcription factor, binds with the TaRE and suppresses TNF-α-mediated P4HA1 expression [106]. In addition, TNF-α can enhance P4HA2 expression at the transcriptional level, and NF-κB/p65 is responsible for TNF-α-mediated P4HA2 upregulation [101].

As a primary factor that drives fibrosis and tumorigenesis, transforming growth factor (TGF)-β can upregulate P4HA1 expression through increasing the binding between the USF1/USF2 and E-box, the motif on human P4HA1 promoter [107]. Suppression of USF1 and/or USF2 results in a significant reduction of P4HA1 transcription [107]. However, cigarette smoking extract can suppress P4HA1 expression via inhibiting the binding between the USF1/USF2 and E-box [107,108].

Although the physiological function of P4HA3 is rarely investigated, zinc finger protein SNAI2 (slug), a transcription factor, can promote P4HA3 transcription in GC [69].

Inhibitors of P4HAs and P4HB

The abovementioned studies implicated that C-P4H may be a potential therapeutic target for cancer patients. In fact, inhibiting enzyme activity or expression of P4HAs or P4HB has been proposed to suppress cancer progression. So far, several pharmacologic inhibitors have been confirmed to have antitumor effects, and some compounds may be candidate inhibitors.

Ethyl 3,4-dihydroxybenzoate

Ethyl 3,4-dihydroxybenzoate (EDHB), a P4HA inhibitor, is commonly used in cell culture experiments [109]. EDHB acts as an analog of the substrate α-ketoglutarate that blocks C-P4H activity, thus effectively inhibiting collagen synthesis [110]. Except inhibiting P4HA activity, EDHB also inhibits other prolyl-hydroxylases, such as HIF-1α prolyl-hydroxylases [109]. A dose of 40 mg/kg EDHB per day in mice was found to decrease primary breast cancer growth, decrease the collagen content of the primary tumor, and significantly reduce lung metastasis without affecting overall body weight [38]. Meanwhile, EDHB also inhibits the proliferation of melanoma cells via inducing apoptosis [111]. However, EDHB also perturbs iron metabolism and affects redox homeostasis. Therefore, the application of EDHB in cancer treatment warrants further investigation.

2-(5-Carboxythiazol-2-yl) pyridine-5-carboxylic acid

Like EDHB, 2-(5-carboxythiazol-2-yl) pyridine-5-carboxylic acid (pythiDC) is an analog of the substrate α-ketoglutarate, which inhibits the activity of C-P4H with a negligible affinity for iron [109,112]. pythiDC can inhibit tumor growth in mice bearing CRC patient-derived xenografts [73]. More importantly, pythiDC neither causes general toxicity nor disrupts iron homeostasis [109]. Thus, pythiDC is a promising candidate for cancer treatment.

Aspirin

Inflammation is an important cause for HCC, and chronic liver inflammation leads to liver cirrhosis and eventually HCC [113]. Aspirin, an anti-inflammatory drug, is widely used in clinics for CRC, ovarian cancer, breast cancer, and prostate cancer [114]. Aspirin can also suppress collagen deposition and subsequent liver tumor growth probably via inhibiting P4HA2 expression [101].

Rutin

P4HB expression is significantly upregulated in a variety of cancers, including HCC, RCC, glioma, GC, and melanoma, and its upregulation correlates with cancer metastasis and invasion. Quercetin-3-rutinoside, also known as rutin (a natural product belonging to the flavonol family), has been reported to possess antitumor and antimicrobialactivities [115]. Rutin inhibits PDI function by binding PDI directly. Such a binding results in a constrained conformation resembling the reduced form of PDI [116]. As a lead inhibitor of PDI, rutin can increase the sensibility of melanoma cells to vemurafenib (the BRAF inhibitor) via targeting P4HB, suggesting that rutin may be used to treat vemurafenib-resistant melanoma patients [33].

Conclusions and Future Directions

This review focused on the clinical and biological significance of C-P4H, paying particular attention to its relevance to carcinogenesis and cancer progression, such as cancer proliferation, invasion, metastasis, angiogenesis, and chemotherapy resistance. As a key enzyme in collagen synthesis, C-P4H can modulate cancer cell behavior via regulating collagen synthesis. In addition, C-P4H also regulates cancer progression through collagen-independent ways, such as modulating tumor cell glycolysis, HIF-1α stability, demethylase activities of TET and JMJ, as well as proline hydroxylation of Carabin or Argonaute 2, but the mechanism remains to be investigated.

Although collagen detected in solid tumors is mostly secreted by cancer associated fibroblasts (CAFs), some tumor cells can also produce collagen [44,117–120]. The functions and underlying mechanisms of C-P4H in CAFs as well as tumor cells during cancer progression remain to be explored.

C-P4H has been identified as a promising prognostic factor or therapeutic target for cancer. Until now, known inhibitors of C-P4H lack clinical relevance in treating cancer patients. Many factors may contribute to this low potency in cancer treatment. C-P4H inhibitors may generate mixed modes of activity, with prohibitive toxicity or intolerable off-target effects [109]. Thus, it will be essential to pursue more effective and safe inhibitors of C-P4H.

Taken together, C-P4H is a potential diagnostic marker and therapeutic target for cancer patients. Further extensive studies are required for their possible clinical applications.

Funding

This work was supported by grants from the National Key R&D program of China (No. 2018YFA0507201 to C.L.) and the National Natural Science Foundation of China (No. 32070147 to C.L.).

Conflict of Interest

The authors declare that they have no conflict of interest.

References