PLOD2 Expression Research Articles

Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

BackgroundMetastatic relapse of prostate cancer after radiotherapy is a significant cause of prostate cancer-related morbidity and mortality. PLOD2 is a mediator of invasion and metastasis that we identified as being upregulated in our highly aggressive radiorecurrent prostate cancer cell line.MethodsPatient dataset analysis was conducted using a variety of prostate cancer cohorts. Prostate cancer cell lines were treated with siRNA, or the drug PX-478 prior to in vitro invasion, migration, or in vivo chick embryo (CAM) extravasation assay. Protein levels were detected by western blot. For RNA analysis, RNA sequencing was conducted on PLOD2 knockdown cells and validated by qRT-PCR.ResultsPLOD2 is a negative prognostic factor associated with biochemical relapse, driving invasion, migration, and extravasation in radiorecurrent prostate cancer. Mechanistically, HIF1α upregulation drives PLOD2 expression in our radiorecurrent prostate cancer cells, which is effectively inhibited by HIF1α inhibitor PX-478 to reduce invasion, migration, and extravasation. Finally, the long non-coding RNA LNCSRLR acts as a promoter of invasion downstream of PLOD2.ConclusionsTogether, our results demonstrate for the first time the role of PLOD2 in radiorecurrent prostate cancer invasiveness, and point towards its potential as a therapeutic target to reduce metastasis and improve survival outcomes in prostate cancer patients.

Systematic characterization of the expression, prognosis and immune characteristics of PLOD family genes in breast cancer.

The expression patterns and prognostic value of Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) family genes in breast cancer remain to be elucidated. The expression levels, prognostic value, and biological function of PLODs were determined using Oncomine, cBioPortal, GEPIA, Timer, UALCAN, PrognoScan, GeneMANIA, Metascape, and breast cancer tissue microarrays. The expressions of PLOD1 and PLOD3 were upregulated in breast cancer tissues, indicating worse clinical stages. High expression levels of PLOD family genes were associated with worse disease-free survival and distant metastasis-free survival, while high expression levels of PLOD1 and PLOD3 were related to worse overall survival in all breast cancer patients. The levels of PLOD family genes were all significantly higher in the age ≤51 y group, HR-negative patients, and triple negative breast cancer (TNBC) patients. They are associated with tumor-infiltrating immune cells (TIICs), including CD4+ T cells, CD8+ T cells, B cells, macrophages, neutrophils, and dendritic cells. According to co-expression gene analysis and functional enrichment, they are associated with protein hydroxylation, collagen biosynthesis and modifying enzymes, collagen metabolism, RNA splicing, extracellular matrix organization, VEGFA-VEGFR2 signaling pathway, and skeletal system development. Immunohistochemistry showed that the expressions of all PLOD family genes were significantly elevated in breast cancer tissues. PLOD1 expression was positively correlated with ER, TNBC status, and tumor grade. PLOD2 expression was positively connected with Ki-67 status. PLOD3 expression was positively related with age and tumor grade. PLOD family genes are novel potential prognostic biomarkers for breast cancer, and targeting PLOD inhibitors might be an effective strategy for breast cancer therapy.

Study on Correlation and Mechanism of Procollagen Lysine 2 Oxoglutarate 5 Dioxygenase on Stem Cell Characteristics and Vasculogenic Mimicry Formation in Esophageal Squamous Cell Carcinoma

In this study, we investigated the significance of the stem cell transcription factor PLOD2 in esophageal cancer to enhance our understanding of its occurrence, development, recurrence, metastasis, and potential treatment. We conducted the following experiments: (1) Immunohistochemistry revealed elevated PLOD2 protein levels in esophageal carcinoma compared to adjacent tissues, with higher levels associated with advanced disease stages and lower differentiation. (2) Immunofluorescence demonstrated increased PLOD2 expression in esophageal cancer cell lines TE-1 and Eca- 109, suggesting a connection to cell differentiation. (3) We successfully transfected fluorescently labeled PLOD2 siRNA into cells, achieving a transfection rate of 67.57% and silencing efficiency exceeding 80%. (4) Following transfection, we observed a decreased proliferation rate in PLOD2 siRNA-treated cells, indicating that PLOD2 reduction can slow esophageal cancer growth. These findings emphasize that PLOD2 plays a crucial role in esophageal cancer pathogenesis, particularly in low cell differentiation maintenance. It could serve as a promising target for inducing differentiation in esophageal cancer and evaluating its malignancy. The high PLOD2 expression in esophageal cancer cell lines suggests the presence of tumor stem cells. Moreover, reducing PLOD2 through RNAi technology slows cell proliferation, suggesting that inhibiting PLOD2 may offer a potential therapeutic approach for esophageal cancer treatment.

Abstract 1274: Investigating PLOD2 as a therapeutic target to overcome metastasis in radiorecurrent prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in males, with 1 in 8 men developing the disease in their lifetime. For PCa, a common treatment strategy is external beam radiation therapy to the prostate. However, when cancer recurs (radiorecurrent PCa), it often behaves aggressively by invading into surrounding organs or spreading distantly. Radiorecurrent PCa metastasis is therefore a significant cause of morbidity and mortality that must be overcome to improve survival of advanced PCa patients. Proteomic analysis revealed the procollagen enzyme Lysyl Hydroxylase 2 (PLOD2) to be upregulated in our radiorecurrent, and highly aggressive, conventionally fractionated DU145 (DU145-CF) PCa cell line. Given its established function as a mediator of invasion in various other cancers, we sought to characterize the role of PLOD2 in the aggressive phenotype of our radiorecurrent PCa cells. Bioinformatic analysis of clinical data revealed PLOD2 genomic amplification to be significantly associated with biochemical recurrence in PCa patients. Upon further examination in vitro, it was revealed that PLOD2 knockdown significantly reduces matrigel invasion and migration in our radiorecurrent DU145-CF cell line, in addition to numerous other PCa cell lines, including primary cells we derived directly from PCa patients. Using the in vivo chick Chorioallantoic Membrane (CAM) model, we confirmed that PLOD2 knockdown significantly reduces the ability of DU145-CF cells to extravasate from the CAM vasculature into the surrounding stroma, a critical step of the metastatic cascade. To explore mechanisms of metastatic cellular reprogramming downstream of PLOD2, RNA sequencing of DU145-CF cells was conducted; a total of 681 genes were discovered to be dysregulated by PLOD2 knockdown, with their functional analysis suggesting changes in cellular metabolism and respiration. Finally, since PLOD2 is known to be regulated by hypoxia-induced protein HIF1α, we explored whether PLOD2 expression could be inhibited by the HIF1α inhibitor, PX-478. Treatment with PX-478 reduced both HIF1α and PLOD2 protein expression, and significantly reduced invasion, migration, and in vivo extravasation in DU145-CF cells, thereby indicating its potential as a pharmacological inhibitor of HIF1α-associated PLOD2 in radiorecurrent PCa. Together, our results demonstrate for the first time the role of PLOD2 in radiorecurrent PCa invasiveness, and point towards its potential as a therapeutic target to reduce metastasis and improve survival outcomes in PCa patients. Citation Format: Gavin Frame, Hon Leong, Roni Haas, Xiaoyong Huang, Jessica Wright, Paul C. Boutros, Thomas Kislinger, Stanley K. Liu. Investigating PLOD2 as a therapeutic target to overcome metastasis in radiorecurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1274.

METTL3 facilitates renal cell carcinoma progression by PLOD2 m6A-methylation under prolonged hypoxia

N6-methyladenosine (m6A) is the most prevalent reversible modification in eukaryotic mRNA, and it plays a critical role in tumor progression. The purpose of this study was to investigate the function and regulatory mechanisms of the methyltransferase METTL3 in renal cell carcinoma (RCC). METTL3 expression was upregulated and predicted a poor prognosis in patients with advanced RCC. METTL3 facilitated the proliferation, migration, and invasion of RCC cells, depending on its methylase activity. METTL3 positively regulated the expression of PLOD2, and both genes were triggered under prolonged hypoxia. Mechanistically, hypoxia-induced the binding of HIF-1α to the METTL3 promoter, which enhanced its transcriptional activity. METTL3-mediated m6A modifications of PLOD2 mRNA at 3’UTR region, promoting the translation of PLOD2 protein. Furthermore, silencing METTL3 impaired RCC progression in vitro. In vivo, administration of highly potent and selective METTL3 inhibitor STM2457 showed anti-tumor effects, whereas AAV9-mediated re-transduction of PLOD2 largely abolished the above phenomenon in a subcutaneous mouse model. These findings reveal that hypoxia and HIF-driven METTL3 transcription promote RCC progression by increasing PLOD2 expression in an m6A-dependent manner, suggesting that METTL3 may serve as a novel pharmaceutical intervention for RCC.

Hypoxia-induced PLOD2 promotes clear cell renal cell carcinoma progression via modulating EGFR-dependent AKT pathway activation

Clear cell renal cell carcinoma (ccRCC) is a type of kidney cancer that is both common and aggressive, with a rising incidence in recent decades. Hypoxia is a key factor that plays a vital role in the tumorigenesis and metastasis of malignancy. However, the precise mechanisms of hypoxia driving ccRCC progression were not totally uncovered. Our study found that hypoxia level was elevated in ccRCC and might be an independent risk factor of prognosis in ccRCC patients. We identified a key protein PLOD2 was induced under hypoxic conditions and strongly associated with poor prognosis in ccRCC patients. When PLOD2 was depleted, the proliferation and migration of ccRCC cells were reduced in vitro and in vivo, while overexpression of PLOD2 had the opposite effect. Mechanically, the study further revealed that PLOD2 was transcriptionally activated by HIF1A, which binds to a specific promoter region of the PLOD2 gene. PLOD2 was also shown to interact with EGFR, leading to the phosphorylation of the receptor. Furthermore, PLOD2 was responsible for binding to the extracellular domain of EGFR, which ultimately activated the AKT signaling pathway, thus promoting the malignant progression of ccRCC. Treatment with the PLOD2 inhibitor Minoxidil significantly suppressed ccRCC progression by inactivating the EGFR/AKT signaling axis. In summary, the findings of this study shed light on the molecular mechanisms behind PLOD2 expression in ccRCC and suggest that it may serve as a potential predictor and therapeutic target for the clinical prognosis and treatment of ccRCC.

Validation of Fourier Transform Infrared Microspectroscopy for the Evaluation of Enzymatic Cross-Linking of Bone Collagen.

Enzymatic cross-linking of the bone collagen is important to resist to crack growth and to increased flexural strength. In the present study, we proposed a new method for assessment of enzymatic cross-link based on Fourier transform infrared (FTIR) microspectroscopy that takes into account secondary structure of type I collagen. Briefly, femurs were collected from sham or ovariectomized mice and subjected either to high-performance liquid chromatography-mass spectrometry or embedded in polymethylmethacrylate, cut and analyzed by FTIR microspectroscopy. FTIR acquisition was recorded before and after ultraviolet (UV) exposure or acid treatment. In addition, femurs from a second animal study were used to compare gene expression of Plod2 and Lox enzymes and enzymatic cross-links determined by FTIR microspectroscopy. We evidenced here that intensities and areas of subbands located at ~1660, ~1680, and ~1690cm-1 were positively and significantly associated with the concentration of pyridinoline (PYD), deoxypyridinoline, or immature dihydroxylysinonorleucine/hydroxylysinonorleucine cross-links. Seventy-two hours exposure to UV light significantly reduced by ~86% and ~89% the intensity and area of the ~1660cm-1 subband. Similarly, 24h of acid treatment significantly reduced by 78% and 76% the intensity and area of the ~1690cm-1 subband. Plod2 and Lox expression were also positively associated to the signal of the ~1660 and ~1690cm-1 subbands. In conclusion, our study provided a new method for decomposing the amide I envelope of bone section that positively correlates with PYD and immature collagen cross-links. This method allows for investigation of tissue distribution of enzymatic cross-links in bone section.

Competitive endogenous RNA-mediated upregulation of PLOD2 expression correlates with poor prognosis and tumor immune infiltration of head and neck squamous cell carcinoma

Competitive endogenous RNA-mediated upregulation of PLOD2 expression correlates with poor prognosis and tumor immune infiltration of head and neck squamous cell carcinoma

Silencing PLOD2 attenuates cancer stem cell-like characteristics and cisplatin-resistant through Integrin β1 in laryngeal cancer

Laryngeal cancer (LC) is an aggressive malignancy resistant to drug treatments. It has been postulated that cancer stem cells (CSCs) persist in a unique population of cancer cells involved in tumor progression and drug-resistance. In the present study, the effects of PLOD2 expression on ordinary and Cisplatin (DDP)-resistance (R) cells were investigated in TU686 and TU138 cells and Xenograft model. Cell viability, invasion and cell apoptosis, CD44 and CD133 expressions, MRP1 and P-gp expressions were measured by CCK-8 assay, Transwell, flow cytometry, immunofluorescence and Western blotting respectively. The results of our study demonstrated that suppressing the expression of PLOD2 could meditate LC stem cell-like features by decrease cell viability and invasion, increase apoptotic rate, decrease CD44 and CD133 expressions via Integrin β1. Meanwhile, the inhibition of PLOD2 expression could decrease P-gp and MRP1expression thus markedly regulate DDP-R LC cells stemness and drug-resistance via Integrin β1. Our findings provided a new rationale for subsequent academic and clinical research on LC drug-resistance.

Pan-Cancer Analyses Reveal Oncogenic and Immunological Role of PLOD2.

Some previous studies have shown that PLOD2 has some value in tumorigenesis. However, the broad significance of PLOD2 has not been discussed in depth. This study was aimed at elaborated and summarized the value of PLOD2 in various tumors. First, we integrated GTEx, The Cancer Genome Atlas and Cancer Cell Line Encyclopedia databases to analyze the expression of PLOD2, and found that it was expressed differently in normal tissues and significantly highly expressed in most tumors compared with normal tissues. Second, our analysis revealed that PLOD2 expression was negatively correlated with the prognosis of several tumors. For gastric cancer, the median overall survival time was significantly higher in the PLOD2 low expression group [HR 0.616 (95%CI 0.442–0.858), p = 0.004]. Third, for tumor immunity, PLOD2 was significantly associated with tumor infiltration, including immune infiltrating cells; immune checkpoint expression; immune microenvironment scores (immune score, stromal score and estimate scores); immunotherapy-related scores (tumor mutational burden, microsatellite instability, tumor neoantigen burden); expression of DNA repair genes Mismatch Repairs and methyltransferase; and enrichment analyses identified PLOD2-associated terms and pathways. Lastly, twenty pairs of gastric cancer and adjacent immunohistochemistry showed that PLOD2 was significantly overexpressed in gastric cancer (p < 0.001). Collectively, PLOD2 played a significant role in tumorigenesis and maybe serve as a potential biomarker for diagnosis and prognosis in cancers.

Control of the extracellular matrix by hypoxic lymphatic endothelial cells: Impact on the progression of lymphedema?

Initial lymphatic vessels do not have a continuous basement membrane. Therefore, the ability of lymphatic endothelial cells (LECs) to produce extracellular matrix (ECM) has received little attention. Untreated lymphedema is a chronic disease that progresses to massive fibrosclerosis in advanced stages. Expansion of the intercellular space and fibrosclerosis cause hypoxia, which also affects the LECs. We studied the expression of genes in human LECs in vitro by RNA sequencing, analyzed the effects of hypoxia (1% O2 ) vs. normoxia (21% O2 ), and focused on ECM genes. LECs express fibrillin-1 and many typical components of a basement membrane such as type IV, VIII, and XVIII collagen, laminin β1, β2, and α4, perlecan, and fibronectin. Under hypoxia, we found significant upregulation of expression of genes controlling hydroxylation of procollagen (PLOD2, P4HA1), and also cross-linking, bundling, and stabilization of collagen fibrils and fibers. Also striking was the highly significant downregulation of elastin expression, whereas fibulin-5, which controls the assembly of tropoelastin monomers, was upregulated under hypoxia. In the dermis from genital lymphedema, we observed significant PLOD2 expression in initial lymphatics. Overall, hypoxia results in the picture of a dysregulated ECM production of LECs, which might be partly responsible for the progression of fibrosclerosis in lymphedema.

PLOD2 Is a Potent Prognostic Marker and Associates with Immune Infiltration in Cervical Cancer.

Background PLOD2 is overexpressed in diverse tumors and plays a vital role in tumorigenesis. However, the prognostic value of PLOD2 in cervical cancer (CESC) remains unclear. Methods PLOD2 expression and CESC patients' survival data were collected from the Oncomine, GEPIA, UALCAN, and Kaplan-Meier Plotter databases; immunohistochemistry (IHC) was used to validate the expression of PLOD2 in CESC; Gene Set Enrichment Analysis was performed using the STRING and DAVID databases; and the correlations between PLOD2 and cancer immune infiltrates were investigated using the TIMER and TISIDB databases. Results We found that the expression level of PLOD2 was increased in various cancers, and meta-analysis in the Oncomine database revealed that PLOD2 was significantly upregulated in CESC compared to that in normal tissues (P < 0.001). In addition, the high expression of PLOD2 was closely related to poor overall survival (OS) and disease-free survival (DFS) in patients with CESC (OS HR = 1.73, P = 0.029; DFS HR = 2.60, P = 0.018). Functional annotations indicated that differentially expressed PLOD2 were primarily related to protein digestion and absorption pathways and to the collagen fibril organization process. Immune infiltration analysis showed that PLOD2 was highly correlated with B cells, CD4+ T cells, T helper type 2 (Th2) cells, and eosinophils in CESC. Conclusion PLOD2 is positively associated with poor prognosis and might be considered a novel diagnostic and prognostic marker for CESC patients.

Targeted Demethylation of the PLOD2 mRNA Inhibits the Proliferation and Migration of Renal Cell Carcinoma.

N6-methyladenosine (m6A) RNA modification is the most common internal mRNA modification in mammals and has been reported to play a key role in gene expression regulation. In this study, we detected a high level of m6A methylation of the PLOD2 3′-untranslated regions (3′UTR) in renal cell carcinoma (RCC). Furthermore, we found that the high expression level of PLOD2 was a prognostic indicator for patients with RCC. A dm6ACRISPR demethylation system was performed to accurately and specifically demethylate 3′UTR of PLOD2 and caused an inactivation of PLOD2 expression. Furthermore, we also performed many in vitro experiments to confirm that PLOD2 exerted tumor promoter effects by promoting tumor proliferation and migration. In conclusion, PLOD2 mRNA demethylated by dCas13b-ALKBH5 might provide a new light on the treatment for RCC.

PLODs are overexpressed in ovarian cancer and are associated with gap junctions via connexin 43

Procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) play important roles in cancer progression, but their role in ovarian cancer remains elusive. In silico analysis of expression of PLODs in ovarian cancer was performed with reproduction of The Cancer Genome Atlas dataset. PLOD-enriched pathways and related gene(s) were validated by immunohistochemistry (IHC) in 80 ovarian cancer tissue blocks and in vivo xenograft murine models. PLODs (PLOD-1, -2, and -3) were overexpressed in ovarian cancer tissue. Overexpression of individual PLODs showed mutual exclusivity. Each of the three PLODs was differentially expressed between normal and cancer tissue of the ovary. PLOD1 was not prognostic, whereas lower PLOD2 and higher PLOD3 expression were associated with worsened prognosis, respectively. Cases with PLOD overexpression showed enrichment in gap junctions. GJA1 (connexin 43) was significantly overexpressed in cases with PLOD overexpression. IHC in tissue showed the strongest positive correlation between PLOD3 and connexin 43 expression, followed by PLOD2. As per Harmonizome, we selected SKOV3 and CAOV3 cell lines based on constitutive high PLOD1 and PLOD2/PLOD3 expression, respectively for in vitro and in vivo modeling. Only knockdown of PLOD3 was significantly associated with decreased GJA1 expression level in both cell lines. IHC in murine xenograft tumors also showed significantly lower connexin 43 in PLOD3-KD SKOV3 tumors. We conclude that PLODs are generally overexpressed in ovarian cancer and each PLOD may be functionally non-redundant. Association between PLOD3 and gap junctions warrants further investigation.

The Collagen-Modifying Enzyme PLOD2 Is Induced and Required during L1-Mediated Colon Cancer Progression.

The overactivation of Wnt/β-catenin signaling is a hallmark of colorectal cancer (CRC) development. We identified the cell adhesion molecule L1CAM (L1) as a target of β-catenin-TCF transactivation in CRC cells. The overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis and liver metastasis, and L1 is exclusively localized in the invasive areas of human CRC tissue. A number of genes are induced after L1 transfection into CRC cells by a mechanism involving the cytoskeletal protein ezrin and the NF-κB pathway. When studying the changes in gene expression in CRC cells overexpressing L1 in which ezrin levels were suppressed by shRNA to ezrin, we discovered the collagen-modifying enzyme lysyl hydroxylase 2 (PLOD2) among these genes. We found that increased PLOD2 expression was required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis and liver metastasis, since the suppression of endogenous PLOD2 expression, or its enzymatic activity, blocked the enhanced tumorigenic properties conferred by L1. The mechanism involved in increased PLOD2 expression by L1 involves ezrin signaling and PLOD2 that affect the SMAD2/3 pathway. We found that PLOD2 is localized in the colonic crypts in the stem cell compartment of the normal mucosa and is found at increased levels in invasive areas of the tumor and, in some cases, throughout the tumor tissue. The therapeutic strategies to target PLOD2 expression might provide a useful approach for CRC treatment.

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFβ1 Activation.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%–70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFβ1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFβ1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFβ1-induced and uninduced PLOD2 expression.

PLOD2 Is Essential to Functional Activation of Integrin β1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas.

SummaryIdentifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers.

Overexpressing PLOD family genes predict poor prognosis in gastric cancer

Procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) are a set of enzymes involved in the hydroxylation of lysine and stabilization of collagen by crosslinks. Previous studies have highlighted that overexpressing PLOD genes were related to the progression, migration and progression of different human cancers. However, the diverse expression patterns and prognostic values of PLOD genes remain to be elucidated in gastric cancer (GC). In this study, we mined the expression and survival data in GC patients through ONCOMINE, UALCAN and Kaplan-Meier Plotter database. STRING portal couple with DAVID was used to establish a functional protein interaction network of PLOD family genes and analyze the GO and KEGG enriched pathways. Differential gene expression correlated with PLOD family genes was identified with LinkedOmics. We found that PLOD1, 2 and 3 were up-regulated in GC patients compared with normal tissues. High expression levels of PLOD1 and PLOD3 were associated with shorter overall survival (OS), first progression (FP) and post progression survival (PPS) while high expression level of PLOD2 was only associated with shorter FP in all GC patients. Specifically, only high PLOD2 expression had significant correlation with shorter OS, FP and PPS in the diffuse type GC patients. Furthermore, combinatorial use of expressions of all PLOD genes was a superior prognostic indicator for GC patients. Pathway analysis confirmed that PLOD family genes mainly participate in regulating the collagen metabolism and extracellular matrix constitution, and the cellular adaptor protein SHC1, which helps to transduce an extracellular signal into an intracellular signal, could be the regulatory module mediating PLOD's effect on GC. Therefore, we propose that individual PLOD genes or PLOD family genes as a whole could be potential prognostic biomarkers for GC.

Abstract B084: PLOD2 is essential to functional activation of integrin beta1 for invasion/metastasis of cancer

Abstract Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in various cancer cells, and its critical role as a stabilizer of ITGB1, enabling ITGB1 to initiate tumor invasion/metastasis, which was demonstrated mainly using SCC cells as a representative example. ITGB1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis in vivo, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of SCC tissues from patients corroborated PLOD2 expression associated with ITGB1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of ITGB1 that prominently regulates tumor invasion and metastasis not only in SCCs but tumors of diverse origins, and it provides important clues engendering novel therapeutics for these intractable cancers. Citation Format: Eisaku Kondo, Ken Saito. PLOD2 is essential to functional activation of integrin beta1 for invasion/metastasis of cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B084. doi:10.1158/1535-7163.TARG-19-B084

An in vitro model to evaluate the properties of matrices produced by fibroblasts from osteogenesis imperfecta and Ehlers-Danlos Syndrome patients

Aim of the studyOsteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts. Materials and methodsFibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled 14C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content. Results and conclusionsOsteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms.