PLOD2 in cancer research

Biomedicine & Pharmacotherapy 90 (2017) 670–676

Available online at

ScienceDirect www.sciencedirect.com

Review

PLOD2 in cancer research Hongzhi Dua , Mao Pangb , Xiaoying Houa , Shengtao Yuanc,* , Li Suna,** a

Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, Jiangsu, China Department of Spine Surgery, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China c Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing, Jiangsu, China b

A R T I C L E I N F O

Article history: Received 8 March 2017 Received in revised form 31 March 2017 Accepted 10 April 2017 Keywords: PLOD2 Lysyl hydroxylase 2 Cancer research Metastasis

A B S T R A C T

Collagen is not only the most abundant protein providing the scaffold for assembly of the extracellular matrix (ECM), but also considered to be the “highway” for cancer cell migration and invasion depending on the different collagen organizations. The accumulation of stabilized collagen is enhanced by different covalent collagen cross-links, lysyl hydroxylases 2 (encoded by the PLOD2 gene) is the key enzyme mediating the formation of the stabilized collagen cross-link. Interestingly, PLOD2 is overexpressed in different cancers and closely related to a poor prognosis. To the best of our knowledge, only the mechanisms of PLOD2 regulated by HIF-1a, TGF-b and microRNA-26a/b have been elaborated. In addition, several pharmacologic inhibitors of PLOD2 have been confirmed to have an anti-metastasis effect. However, there have been no reviews about PLOD2 in cancer research published thus far. In brief, this review about PLOD2 will describe the function, regulatory mechanism, and the inhibitors of PLOD2 in cancer, suggesting the credible clinical evaluation of a prognostic signature in pathological examination and the possible development of therapeutic strategies targeting PLOD2 in the future. © 2017 Published by Elsevier Masson SAS.

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Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PLOD2 in different cancers . . . . . . . . . . . . . . . . . Hepatocellular carcinoma . . . . . . . . . . . . 2.1. Breast cancer . . . . . . . . . . . . . . . . . . . . . . 2.2. Sarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Other cancers . . . . . . . . . . . . . . . . . . . . . 2.4. PLOD2 in tumour microenvironment (TME) . . . CAFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. 3.2. Stellate cells . . . . . . . . . . . . . . . . . . . . . . Other stromal cells . . . . . . . . . . . . . . . . . 3.3. The regulatory mechanisms of PLOD2 in cancer HIF-1a . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. MicroRNA-26a/b . . . . . . . . . . . . . . . . . . . 4.2. 4.3. TGF-b . . . . . . . . . . . . . . . . . . . . . . . . . . . Other potential regulations . . . . . . . . . . 4.4. The inhibitor of PLOD2 . . . . . . . . . . . . . . . . . . . . Minoxidil . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. 5.2. Natural products . . . . . . . . . . . . . . . . . . . Other candidate inhibitors . . . . . . . . . . . 5.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing, China. ** Corresponding author at: Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, No. 24, Tongjiaxiang, Nanjing, China. E-mail addresses: [email protected] (S. Yuan), [email protected] (L. Sun). http://dx.doi.org/10.1016/j.biopha.2017.04.023 0753-3322/© 2017 Published by Elsevier Masson SAS.

H. Du et al. / Biomedicine & Pharmacotherapy 90 (2017) 670–676

Availability of data and material Conflicts of interest statement . Acknowledgements . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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1. Background Mounting evidence currently suggests that the progression of tumour is determined not only by tumour cells, but also by the tumour microenvironment (TME), while previous studies regarding tumour metastasis have primarily focused on the adhesion and migration ability of cancer cells themselves. Moreover, as the chief component of the TME, the extracellular matrix (ECM), plays a significant role in tumour progression, including differentiation, proliferation, migration, adhesion and survival, especially in tumour metastasis [1–3]. Meanwhile, collagen is not only considered to be the most abundant protein providing the scaffold for ECM assembly but is also considered to be the “highway” for cancer cell migration and invasion [4,5]. Collagen is no longer thought to provide a barrier for migration and invasion but is now considered to promote metastasis based on different collagen organizations [6]. Evidence from multiple types of human cancers suggests that the accumulation of stabilized collagen is enhanced by different covalent collagen cross-links [7,8]. As previously reported, lysyl hydroxylases 2 (LH2, encoded by the PLOD2 gene) is the key enzyme mediating the formation of stabilized collagen cross-links [9]. PLOD2 is one member of the PLOD family (PLOD1, PLOD2, and PLOD3). Only PLOD2 plays the key role in formation of stabilized collagen cross-links by hydroxylation of lysyl residues [10]. Gainand loss-of-function studies showed that LH2 hydroxylated telopeptidyl lysine residues on collagen, shifted the tumour stroma toward higher levels of hydroxylysine aldehyde–derived collagen cross-links (HLCCs), lower levels of lysine aldehyde– derived cross-links (LCCs), increased tumour stiffness, and enhanced tumour cell invasion and metastasis [4,5]. PLOD2 was first reported about the activity of LH2 in the liver with hepatic injury, in 1974 [11]. In 1996, PLOD2 was first reported in breast cancer research by Smith [12]. In recent years, an increasing number of articles on PLOD2 in cancer research have been published and cited (Fig. 1A and B). Thus far, there have been no reviews on PLOD2 in cancer research. In this article, we will describe the function and mechanisms of PLOD2 in different cancers and explore the potential pharmacologic inhibitors of PLOD2 for their anti-metastatic effect, demonstrating the credible clinical evaluation of a prognostic signature in the pathological

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examination and suggesting the possible future development of therapeutic strategies targeting the TME. 2. PLOD2 in different cancers According to the Cancer Cell Line Encyclopedia (CCLE) project, PLOD2 has been reported in many different cancers as shown in Fig. 2A. Furthermore, the expression of PLOD2 in various cancers is strikingly different. Further database analysis demonstrates that PLOD2 expression is higher in adenocarcinoma tissues than in normal tissues via the OncomineTM database in several cancers such as lung, breast, liver, pancreas and so on (Fig. 3A–D). In fact, sarcomas [7], bladder cancer [13], renal cell carcinoma [14], glioblastoma [15], cervical cancer [16], oral carcinoma [17], bone metastasis [18] and other cancers also significantly overexpressed PLOD2, as previously reported. The overexpression of PLOD2 is closely related to poor prognosis based on Kaplan-Meier plotter, as shown in Fig. 3G and H. Otherwise, when the PLOD2 expression is not different in gastric adenocarcinoma tissues versus normal tissues, it consistently has no relation with prognosis in gastric cancer (Fig. 3F and I). In brief, PLOD2 may be a biomarker for poor prognosis in several cancers. 2.1. Hepatocellular carcinoma PLOD2 was first confirmed as a novel prognostic factor in hepatocellular carcinoma (HCC), in 2011 [19]. When compared with the low-expression group, the disease-free survival time in the high PLOD2 expression group of HCC patients is significantly shorter. PLOD2 expression is significantly correlated with tumour size and macroscopic intrahepatic metastasis among all the clinico-pathological factors. In univariate analysis, six prognostic factors (macroscopic intrahepatic metastasis, tumour multiplicity, microscopic portal invasion, histological grade, macroscopic intrahepatic metastasis, and PLOD2 expression) are significant for disease-free survival. In brief, PLOD2 is identified as a significant, independent factor of poor prognosis. Furthermore, the study also preliminarily shows that PLOD2 expression could be regulated by hypoxia, but the mechanism of regulation is still unknown.

Fig. 1. Published articles on PLOD2 in cancer research. (A). The number of published papers about PLOD2 in cancer research. (B). The number of cited papers about PLOD2 on cancer research. The number is based on Web of ScienceTM Core Collection. Topic = ((TOPIC: (PLOD2) AND TOPIC: (Cancer)) OR (TOPIC: (Lysyl hydroxylase 2) AND TOPIC: (Cancer))). (http://apps.webofknowledge.com/).

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Fig. 2. PLOD2 expression in different cancer cell lines. The data are from the Cancer Cell Line Encyclopedia (CCLE) project (https://portals.broadinstitute.org/ccle/home).

Fig. 3. PLOD2 is a prognostic signature for survival in human cancer. (A–F). PLOD2 expression is higher in adenocarcinoma tissues (show in red) than that in normal tissues (show in blue) via the OncomineTM database in lung cancer (A), breast cancer (B), liver cancer (C), pancreatic cancer (D), colon cancer (E) and gastric cancer (F). (G–I). High PLOD2 expression indicated significantly shorter overall survival (OS) than low PLOD2 expression in lung cancer (G), breast cancer (H) and gastric cancer (I). The data are from Kaplan-Meier Plotter (http://kmplot.com/analysis).

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2.2. Breast cancer The function and mechanism of PLOD2 in breast cancer have been thoroughly explored. In clinical, aligned collagen is recognized as a prognostic signature for survival in human breast carcinoma [20], while PLOD2 is now confirmed as the primary key enzyme in the formation of aligned collagen [21]. The clinical data analysis shows that the PLOD2 expression level is significantly higher in breast cancer tissue than in normal tissue and that high PLOD2 expression is significantly associated with decreased disease-specific survival [21]. Obviously, PLOD2 expression is specifically prognostic in breast cancer. Moreover, PLOD2 expression is also confirmed to be regulated by hypoxia-inducible factor 1a (HIF-1a). PLOD2 has been shown to be critical for fibrillar collagen formation by breast cancer cells; it also increases tumour stiffness, and is required for metastasis to lymph nodes and lungs [21]. 2.3. Sarcoma Sarcoma is another cancer for which the function and mechanism of PLOD2 has fully demonstrated. Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, and approximately 40% of these patients ultimately succumb to lethal metastases [22]. As previously reported [7], it is suggested that hypoxia controls sarcoma metastasis through a novel mechanism by which HIF-1a enhances the expression of PLOD2. The report also shows that loss of PLOD2 expression disrupted collagen modification, cell migration, pulmonary metastasis, but not primary tumour growth. But, pharmacologic inhibition of PLOD2 enzymatic activity suppresses metastases. Therefore, it concludes that PLOD2 may be a novel therapeutic target in sarcomas and successful inhibition of PLOD2 could reduce tumour cell dissemination. 2.4. Other cancers In bladder cancer and renal cell carcinoma, Kaplan–Meier analysis also shows that patients with high PLOD2 expression have significantly shorter overall survival (OS) than those with low PLOD2 expression, whereas knockdown of PLOD2 could markedly inhibit the migration and invasion activity [13,14]. In glioblastoma

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[15], cervical cancer [16] and oral carcinoma [17], PLOD2 is highly expressed, but the correlation with prognosis is largely unexplored. Moreover, we have confirmed the function and mechanisms of PLOD2 in lung cancer in vitro, in vivo and clinically (papers will be submitted). 3. PLOD2 in tumour microenvironment (TME) The ECM is not only from the cancer cells themselves but also from the stromal cells, especially cancer associated fibroblasts (CAFs) [23] and stellate cells [24] in the TME. The published reports suggest that CAFs and stellate cells could induce the collagen crosslink switch via PLOD2, promoting tumour cell invasion and migration. In fact, in the TME, the other stromal cells such adipocytes, macrophages [25,26] and human umbilical vein endothelial cells (HUVEC) [27] may similarly participate in the ECM for metastasis through PLOD2 (Fig. 4A); their function and mechanism should be further confirmed. 3.1. CAFs CAFs are the most representative mesenchymal cells in the TME. CAFs play critical roles in the regulation of tumour fibers, immunosuppression, angiogenesis, and metastasis [28,29]. CAFs lead collectively migrating tumour cells by realigning impeding collagen fibers through proteolytic and matrix remodeling, creating tracks for tumour cells movement; the realigned collagen fibers within the track walls have acquired a certain degree of stability through collagen cross-linking [30]. It has been reported that PLOD2 is observed in melanoma-, lung adenocarcinoma-, and multiple myeloma-associated fibroblasts, as well as in hepatocellular carcinoma-associated fibroblasts [31,32]. According to Kurie’s research [23], CAFs induce a collagen cross-link switch through PLOD2 in tumour stroma to influence the invasive properties of tumour cells. PLOD2, which drives higher hydroxylysine aldehyde– derived collagen cross-link (HLCC) formation, is highly expressed in CAFs, and PLOD2 depletion abrogates the ability of CAFs to promote tumour cell invasion and migration. 3.2. Stellate cells Currently, it is a well-established fact that activated pancreatic stellate cells (PSCs) are responsible for the production of the paracrine growth factors, proteolytic enzymes, and ECM

Fig. 4. PLOD2 in tumour microenvironment (TME). The stromal cells including CAF, stellate cells, adipocytes, macrophages and HUVEC could induce the collagen cross-link switch through PLOD2, thereby promoting tumour cell invasion and migration.

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components, which promotes proliferation, migration, and invasion of cancer cells [33,34]. Microarray analysis shows that PLOD2 in PSCs is the gene that potentially regulates ECM fibre architecture under hypoxia [24]. Moreover, knockdown of PLOD2 in PSCs blocks parallel fibre architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that PLOD2 expression in PSCs create a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM.

HIF could regulate PLOD2 in hepatocellular carcinoma [19], breast cancer [21], PSCs [24] and sarcomas [7]. In hypoxic TME, HIF-1a activates transcription of the PLOD2 genes encoding procollagen lysyl hydroxylase that is required for the biogenesis of collagen, which is a major constituent of the ECM. The mechanism of PLOD2 regulated by HIF-1a has been fully confirmed in vitro, in vivo and clinically [7,19,21]. Therefore, specific inhibitors of HIF-1a or PLOD2 may block collagen fibre biogenesis by both cancer cells and stromal cells.

3.3. Other stromal cells

4.2. MicroRNA-26a/b

Fat-conditioned medium could induce PLOD2 expression through prostaglandin F-2a in fibrosis disease. Macrophageconditioned medium also increases collagen deposition, proliferation and gene expression of PLOD2 [25], possibly through hypoxia/ IL-10 [26]. Similarly, hypoxia could regulate PLOD2 expression in HUVEC [35].

Presently, the regulation of PLOD2 by microRNA-26a/b has been confirmed in breast cancer, bladder cancer and renal cell carcinoma [13,14,37]. MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein-coding genes by binding to the 30 untranslated region (UTR) of the target mRNA and inhibiting transcription, while miRNAs are aberrantly expressed in various human cancers and have played significant roles in human oncogenesis and metastasis [38,39]. In silico analyses suggest that PLOD2 is a promising candidate target gene of microRNA-26a/b [13,14]. The restoration of miR-26a-5p and miR-26b-5p results in down-regulation of PLOD2, whereas knockdown of PLOD2 inhibits migration and invasion but not proliferation. Besides, the regulation of PLOD2 by microRNA-26a/b has been confirmed in clinical samples [13,14,37].

4. The regulatory mechanisms of PLOD2 in cancer The function of PLOD2 in the collagen cross-link switch, cell invasion and migration is fully identified for metastasis. Therefore, PLOD2 is confirmed as a novel prognostic factor in several cancers, especially in hepatocellular carcinoma, breast cancer, sarcomas, bladder cancer and renal cell carcinoma [7,13,14,19,21]. So far, only the mechanisms of PLOD2 regulated by HIF-1a, TGF-b and microRNA-26a/b have been elaborated (Fig. 5A). But the regulatory mechanism of PLOD2 is largely unexplored. 4.1. HIF-1a The mechanism of PLOD2 regulated by HIF-1a is first and widely confirmed in cancer research [36]. As reported previously,

4.3. TGF-b Transforming growth factor-b (TGF-b) is recognized as the key regulator of PLOD2 for the formation of collagen crosslinks in fibrosis [40]. In fibroblasts, it has been shown that TGF-b induces a SP1- and SMAD3-dependent recruitment of histone modifying enzymes to the PLOD2 promoter for the expression [41]. The NF-k B

Fig. 5. The regulatory mechanisms of PLOD2 in cancer. To the best of our knowledge, only the mechanisms of PLOD2 regulated by HIF-1a, TGF-b and microRNA-26a/b have been elaborated.

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pathway plays an important role during the induction of PLOD2 expression by TGF-b, in myofibroblast biology [42]. The correlation between TGF-b and PLOD2 in cancer research has not been fully explored. Therefore, CAF as a subtype of fibroblast may be regulated by TGF-b in TME. Since TGF-b is a significant factor in cancer progression including metastasis, PLOD2 also may be induced by TGF-b in cancer pathology. 4.4. Other potential regulations In fact, according to previous reports, some other candidate mechanism may also be involved in the regulation of PLOD2. Bank’s research showed that LH2 activity is somehow dependent on peptidyl-prolyl cis-trans isomerase (PPIase) FKBP65, while the mechanism in cancer has not been confirmed [43]. Research on human leukocyte antigen (HLA) ligands has also suggested that PLOD2 may be the target gene in renal cell carcinoma [44]. Prostaglandin F-2a can regulate PLOD2 by fat-conditioned medium [45]. Research shows that Ras homolog ge1ne family member A (RhoA) also induces PLOD2 expression [46]. However, these regulatory mechanisms of PLOD2 need to be further confirmed. In addition, we have confirmed several novel mechanisms of PLOD2 in breast cancer and lung cancer in vitro, in vivo and clinically (papers will be submitted). 5. The inhibitor of PLOD2 All above, it is fully confirmed that PLOD2 may be a potential therapeutic target for metastasis. Therefore, the inhibitor of PLOD2 could not only act on the cancer cells but also affect the whole TME, and thus control the metastasis. Thus far, several pharmacologic inhibitors have been confirmed to have an anti-metastasis effect, whereas some compounds may be candidate inhibitors.

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expression; nevertheless, their anti-metastatic effect is still unknown. 6. Conclusion On the basis of the reports mentioned here, it sufficiently suggests that PLOD2 may be a novel prognostic factor, as it is overexpressed in many cancers and is a significant factor for disease-free survival. In the TME, stromal cells are also equally significant for the “highway” remodeling through the PLOD2induced collagen cross-link switch. Although several regulatory mechanism of PLOD2 have been fully confirmed, most mechanisms in cancer research should be further explored. Further, even though several pharmacologic inhibitors of PLOD2 have been confirmed to have an anti-metastatic effect, regardless of the inhibition of the target, the pharmacodynamics effect and the effective constituent should be further studied. In brief, in this review about PLOD2, it is concluded that PLOD2 might be a novel prognostic factor. Importantly, the pathological examination of tumour collagen or PLOD2 may be a credible prognostic signature in clinical evaluation, suggesting the possible development of therapeutic strategies targeting collagen in the future. Availability of data and material The datasets generated during the current study are available in the Web of ScienceTM Core Collection repository (http://apps. webofknowledge.com), the Cancer Cell Line Encyclopedia (CCLE) project (https://portals.broadinstitute.org/ccle/home), OncomineTM database (https://www.oncomine.org) and Kaplan-Meier Plotter (http://kmplot.com/analysis). Conflicts of interest statement There is no conflict of interest for submitting this manuscript in all the authors listed.

5.1. Minoxidil Acknowledgements Minoxidil is the only inhibitor of PLOD2 that has been fully confirmed by previous reports [7,47,48]. Interestingly, reports show that minoxidil could suppress the expression of PLOD2, inhibit the cancer migration, and reverse the collagen cross-link switch, resulting in the anti-metastasis effect in vitro and in vivo [7,47]. Therefore, these data show the potential usefulness of minoxidil as a treatment for premetastatic carcinoma. 5.2. Natural products Natural products are becoming increasingly accepted as the potential mode for drug development [49]. Berberine suppresses the expression of PLOD2 and inhibits pulmonary metastasis in melanoma [50]. Amentoflavone [51] and beta-carotene [52] can also decrease PLOD2 expression, and inhibit the tumour metastasis. Meanwhile, some extracts from herbs such as Calendula officinalis (L) flowers [53], and Biophytum seusitivum (L.) DC [54] may exhibit anti-metastatic effect through PLOD2. Nonetheless, the inhibition of target, the pharmacodynamics effect and the effective constituent should be further explored. 5.3. Other candidate inhibitors As previously reported [55], b-aminopropionitrile and homocysteine are known to inhibit stable matrix formation and PLOD2

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