Secreted Phosphoprotein 1 Expression Research Articles

Secreted phosphoprotein 1 as a potential prognostic and immunotherapy biomarker in multiple human cancers

ABSTRACT Secreted phosphoprotein 1 (SPP1) is involved in immune regulation, cell survival, and tumor progression. Studies have demonstrated that SPP1 plays an important role in certain individual tumors. However, the expression profile and oncogenic features of SPP1 in diverse cancers are remaining unknown. Therefore, we performed a comprehensive analysis using The Cancer Genome Atlas (TCGA) database. Raw data of 33 cancer types were download from the University of California Santa Cruz (UCSC) Xena website. The expression of SPP1 and its relationship with tumor prognosis, immune invasion, tumor microenvironment, and immunotherapy were analyzed using the R language. The function analysis was conducted using Gene Set Enrichment Analysis (GSEA). The oncogenic features of SPP1 was validated by wound-healing assay and EdU staining assay. SPP1 highly expressed in most cancers. The expression of SPP1 was significant related to prognosis, tumor mutation burden (TMB), microsatellite instability (MSI), and immune checkpoint genes, suggested that SPP1 plays an essential role in the tumor immune microenvironment and immune cell infiltration. The immune/stromal scores correlated positively with the SPP1 expression, and the relationship was affected by tumor heterogeneity and immunotherapy. In addition, SPP1 could predict the response of tumor immunotherapy. Functional analysis revealed the SPP1-associated terms and pathways. Finally, SPP1 significantly elevated cell proliferation and migration in A549, Huh7, HT-29, A2780 tumor cell lines. In conclusion, this study indicated that SPP1 involved in tumorigenesis, tumor progression, and regulated tumor immune microenvironment, revealing SPP1 might be a potential target for evaluating prognosis and immunotherapy in multiple cancers.

SPP1 Regulates Radiotherapy Sensitivity of Gastric Adenocarcinoma via the Wnt/Beta-Catenin Pathway.

Purpose Radiotherapy has been widely applied for the treatment of locally advanced and metastatic gastric adenocarcinoma (GAC). The aberrant expression of secreted phosphoprotein 1 (SPP1) is involved in radiosensitivity in a variety of cancers. The present study aims to characterize the clinical significance of SPP1 expression in GAC and its role and underlying mechanism of radiosensitivity. Methods The SPP1 expression in GAC tissues and pericarcinomatous tissues was determined by QRT-PCR and immunohistochemistry, and the SPP1 expression in GAC cell lines (BGC823, AGS, and SGC7901) and normal human gastric epithelial cell line (GES-1) was determined by western blot. T-test, one-way ANOVA, Cox regression model, and Kaplan–Meier plotter were applied to further assess the association between SPP1 expression and the prognosis of the patients with GAC. After irradiation and transfection with si-SPP1 combined with or without Wnt/β-catenin pathway inhibitor (XAV939), western blot, transwell, flow cytometry, and TOP-flash reporter assay were applied to detect DNA damage, invasion, apoptosis, cell cycle, and activation of Wnt/β-catenin pathway, respectively. Results SPP1 mRNA and protein levels in GAC tissues were both dramatically higher than those in pericarcinomatous tissues. SPP1 overexpression was positively associated with tumor size, nodal status, and histological grade of GAC patients. SPP1 overexpression, depth of invasion, and nodal status were independent prognostic factors for the patients. High SPP1 expression was negatively related to the overall survival in patients with GAC. We found that SPP1 knockdown enhanced the radiosensitivity of GAC cell lines (AGS and SGC7901). Increasing H2AX phosphorylation, apoptosis and G2/M phase arrest, and decreasing invasion were observed after the administration of si-SPP1 and irradiation. Radiosensitivity of SPP1 was mainly dependent on the Wnt/β-catenin signal pathway. XAV939 could enhance these phenomena induced by irradiation combined with SPP1 knockdown. Conclusion This study demonstrates that SPP1 suppresses Wnt/β-catenin signaling to enhance the radiosensitivity of GAC via inhibiting invasion and accelerating DNA damage, G2/M phase arrest, and apoptosis.

The Correlation Between SPP1 and Immune Escape of EGFR Mutant Lung Adenocarcinoma Was Explored by Bioinformatics Analysis.

BackgroundImmune checkpoint inhibitors have achieved breakthrough efficacy in treating lung adenocarcinoma (LUAD) with wild-type epidermal growth factor receptor (EGFR), leading to the revision of the treatment guidelines. However, most patients with EGFR mutation are resistant to immunotherapy. It is particularly important to study the differences in tumor microenvironment (TME) between patients with and without EGFR mutation. However, relevant research has not been reported. Our previous study showed that secreted phosphoprotein 1 (SPP1) promotes macrophage M2 polarization and PD-L1 expression in LUAD, which may influence response to immunotherapy. Here, we assessed the role of SPP1 in different populations and its effects on the TME.MethodsWe compared the expression of SPP1 in LUAD tumor and normal tissues, and in samples with wild-type and mutant EGFR. We also evaluated the influence of SPP1 on survival. The LUAD data sets were downloaded from TCGA and CPTAC databases. Clinicopathologic characteristics associated with overall survival in TCGA were assessed using Cox regression analysis. GSEA revealed that several fundamental signaling pathways were enriched in the high SPP1 expression group. We applied CIBERSORT and xCell to calculate the proportion and abundance of tumor-infiltrating immune cells (TICs) in LUAD, and compared the differences in patients with high or low SPP1 expression and wild-type or mutant EGFR. In addition, we explored the correlation between SPP1 and CD276 for different groups.ResultsSPP1 expression was higher in LUAD tumor tissues and in people with EGFR mutation. High SPP1 expression was associated with poor prognosis. Univariate and multivariate cox analysis revealed that up-regulated SPP1 expression was independent indicator of poor prognosis. GSEA showed that the SPP1 high expression group was mainly enriched in immunosuppressed pathways. In the SPP1 high expression group, the infiltration of CD8+ T cells was lower and M2-type macrophages was higher. These results were also observed in patients with EGFR mutation. Furthermore, we found that the SPP1 expression was positively correlated with CD276, especially in patients with EGFR mutation.ConclusionSPP1 levels might be a useful marker of immunosuppression in patients with EGFR mutation, and could offer insight for therapeutics.

Upregulation of SPP1 Is a Marker for Poor Lung Cancer Prognosis and Contributes to Cancer Progression and Cisplatin Resistance.

The chemoresistance of lung cancer is a significant contributor to its high mortality and morbidity rate. There is an urgent need to identify differentially expressed genes in lung cancer patients with a poor prognosis to develop effective means to overcome drug resistance in subsequent treatment. In this study, we identified the secreted phosphoprotein 1 (SPP1) as a potential gene associated with a poor diagnosis of lung cancer patients using the Cancer Genome Atlas analysis, which suggested that the expression of SPP1 in tumor tissues was significantly higher than normal tissues. The high expression of SPP1 was also correlated with tumor grade and poor clinical prognosis. To understand the roles of SPP1 and the DNA methyltransferase 1 (DNMT1), which regulated SPP1 expression, in affecting cell viability, migration and invasion, SPP1 and DNMT1 were overexpressed in the human lung cancer A549 and NCI-446 cells, followed by analyzing cell viability, migration and invasion. We showed that SPP1 promoted the proliferation, migration and invasion of lung cancer cells, and increased the resistance of lung cancer to the chemotherapeutic drug cisplatin. Knocking down SPP1 in cells restored sensitivity to cisplatin. Further, A549 cells without SPP1 overexpression demonstrated lower tumor growth rate than SPP1 overexpression cells using the xenograft tumor mouse model. High expression of SPP1 in lung cancer tumor tissue was caused by the reduced methylation level of its promoter region mediated by DNMT1. Our data suggested that SPP1 can be used as a marker for highly malignant lung cancer and targeting SPP1 may be a potential lung cancer treatment strategy.

SPP1 Promotes Enzalutamide Resistance and Epithelial-Mesenchymal-Transition Activation in Castration-Resistant Prostate Cancer via PI3K/AKT and ERK1/2 Pathways.

The bottleneck arising from castration-resistant prostate cancer (CRPC) treatment is its high metastasis potential and antiandrogen drug resistance, which severely affects survival time of prostate cancer (PCa) patients. Secreted phosphoprotein 1 (SPP1) is a cardinal mediator of tumor-associated inflammation and facilitates metastasis. In our previous study, we firstly revealed SPP1 was a potential hub signature for predicting metastatic CRPC (mCRPC) development. Herein, we integrated multiple databases to explore the association of SPP1 expression with prognosis, survival, and metastatic levels in CRPC progression and investigated SPP1 expression in PCa tissues and cell lines. Next, PCa cell lines with overexpression or depletion of SPP1 were established to study the effect of SPP1 on enzalutamide sensitivity and adhesion and migration of prostate cancer cell lines and further explore the underlying regulatory mechanisms. Bioinformatics analysis, polymerase chain reaction (PCR), immunohistochemical staining, and western blot results suggested SPP1 upregulation had strong relationship with the malignant progression of CRPC and enzalutamide resistance. SPP1 knockdown enhanced enzalutamide sensitivity and repressed invasion and migration of prostate cancer cells. Importantly, upregulating SPP1 promoted, while silencing SPP1 attenuated epithelial-mesenchymal-transition (EMT). Our results further demonstrated that SPP1 overexpression maintains the activation of PI3K/AKT and ERK1/2 signaling pathways. Overall, our findings unraveled the functional role and clinical significance of SPP1 in PCa progression and help to discover new potential targets against mCRPC.

Steroid regulation of secreted phosphoprotein 1 (SPP1) expression in ovine endometrium.

Secreted phosphoprotein 1 (SPP1) is an extracellular matrix glycoprotein that is highly expressed at the maternal-fetal interface and is a critical mediator of embryo implantation. The objectives of this study were to examine the spatial and temporal cyclical expression patterns and steroid regulation of SPP1 mRNA and protein in ovine endometrium, which may be further indicative of their functionality in embryo implantation. Uterine tissue was obtained following hysterectomy from ovariectomised ewes treated with ovarian steroids. In parallel, invitro culture of endometrial cells was used to investigate the effects of ovarian steroids on SPP1 expression in endometrial and luminal epithelial (LE) cells. A significant sustained mid-luteal phase increase in SPP1 mRNA in intercaruncular regions of the endometrium was observed, indicating that glandular epithelium is likely to be the primary source of SPP1 production. This increase in SPP1 was induced by progesterone treatment and was shown at the protein level by immunohistochemistry analysis. Similarly, treatment of stromal cells with 10ng mL-1 progesterone or in combination with 1ng mL-1 oestradiol significantly increased SPP1 expression (P<0.05). Collectively, expression levels of SPP1 are cycle-dependent and peak in the progesterone-dominant luteal phase. They are dependent on the interaction of uterine LE and stromal cells and may involve paracrine signalling by progesterone receptor-positive stromal cells.

The Significance of Secreted Phosphoprotein 1 in Multiple Human Cancers.

Malignant tumor represents a major reason for death in the world and its incidence is growing rapidly. Developing the tools for early diagnosis is possibly a promising way to offer diverse therapeutic options and promote the survival chance. Secreted phosphoprotein 1 (SPP1), also called Osteopontin (OPN), has been demonstrated overexpressed in many cancers. However, the specific role of SPP1 in prognosis, gene mutations, and changes in gene and miRNA expression in human cancers is unclear. In this report, we found SPP1 expression was higher in most of the human cancers. Based on Kaplan-Meier plotter and the PrognoScan database, we found high SPP1 expression was significantly correlated with poor survival in various cancers. Using a large dataset of colon adenocarcinoma (COAD), head and neck cancer (HNSC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) patients from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases, this study identified 22 common genes and 2 common miRNAs. GO, and KEGG paths analyses suggested that SPP1 correlated genes were mainly involved in positive regulation of immune cell activation and infiltration. SPP1-associated genes and miRNAs regulatory networks suggested that their interactions may play a role in the progression of four selected cancers. SPP1 showed significant positive correlation with the immunocyte and immune marker sets infiltrating degrees. All of these data provide strong evidence that SPP1 may promote tumor progress through interacting with carcinogenic genes and facilitating immune cells’ infiltration in COAD, HNSC, LUAD, and LUSC.

Impact of birth weight and neonatal nutritional interventions with micronutrients and bovine colostrum on the development of piglet immune response during the peri-weaning period

Immune system development of piglets is influenced by birth weight and colostrum and milk intake. Moreover, the dam transfer to piglets of vitamins A and D and copper, which play important role in immunity, is limited during lactation. In this study, we evaluated the potential of maternal and neonatal supplementations with vitamins A and D and copper, with or without neonatal supplementation of bovine colostrum (BC), to modulate the immune system development of low birth weight (LBW) and high birth weight (HBW) piglets during the peri-weaning period. Litters from 23 control sows (CONT) were assigned to one of the following treatments: 1) control (C); 2) oral administration at 2 and 8 days (d) of age of retinol-acetate, 25-hydroxyvitamin D and CuSO4 and exposure to UVB light for 15 min every second day from d 5 to d 21 (ADCu); 3) oral administration of dehydrated BC (4 g/d) from d 5 to d 10 (BC); 4) ADCu + BC. This experimental design was repeated with 24 sows fed extra daily supplements of 25-hydroxyvitamin D (4,000 IU), β-carotene (30,000 IU) and Cu-yeast (equivalent 45 mg of Cu) from 90 d of gestation until weaning at d 21 (SUPPL). Within each litter, 2 LBW and 2 HBW piglets were euthanized at d 16 and d 23 in order to characterize leukocyte subsets in mesenteric lymph nodes (MLN) and blood by flow cytometry, and to measure gene expression in the MLN and jejunal mucosa by qPCR. At d 16, results revealed that the percentages of γδ and cytotoxic T lymphocytes were significantly reduced in LBW compared to HBW piglets. The jejunal expression of interleukin (IL) 22 was also up-regulated, along with MLN expression of C-C Motif Chemokine Ligand 23, bone morphogenetic protein 2 and secreted phosphoprotein 1 (SPP1), whereas jejunal expression of tumor necrosis factor α was decreased in LBW piglets. At d 23, LBW piglets showed lower amounts of γδ T lymphocytes, higher percentages of CD3− and CD3−CD8α+CD16+ leukocytes (which include Natural killer cells) and lower jejunal expression of IL18. Furthermore, supplementation with BC increased the blood percentage of CD3−CD16+ leukocytes and reduced jejunal IL5 and MLN IL15 expression whereas supplementation with ADCu + BC increased jejunal TNF superfamily 13B and MLN SPP1 expression. Our results suggest that immune system development after birth differed between LBW and HBW piglets and that early dietary supplementation with BC and ADCu has the potential to modulate development of immune functions.

Analysis of Expression and Its Clinical Significance of the Secreted Phosphoprotein 1 in Lung Adenocarcinoma.

ObjectiveTo explore the expression of secreted phosphoprotein 1 (SPP1) in lung adenocarcinoma (LUAD), and evaluate its relationship with clinicopathological characteristics and prognosis of LUAD, and analyze the advantages of SPP1 as a potential prognostic marker in LUAD.MethodsThe expression of SPP1 in normal lung tissue and LUAD was analyzed from the Cancer Cell Line Encyclopedia (CCLE), Gene Expression Omnibus (GEO), and Human Protein Atlas (HPA) databases. GSE68465 was used to explore the relationship between the SPP1 expression and clinicopathological characteristics and the prognosis of LUAD patients. The relationship between SPP1 and immune infiltration in LUAD was analyzed by the Tumor Immune Estimation Resource (TIMER) database. Gene enrichment analysis was performed in GSEA. The Cancer Genome Atlas (TCGA)-LUAD data was used to verify the results.ResultsIn the cell line level, non-small cell lung cancer ranked ninth among cancer cell lines based on SPP1 expression. In the messenger RNA (mRNA) and protein levels, SPP1 expression was higher in LUAD tissues than that in normal control. SPP1 expression was related to gender, N stage, histological grade, and progression or relapse. In men, SPP1 expression were higher compared to that in women. The higher the N stage, the higher the SPP1 expression level. As LUAD progresses or relapses, SPP1 expression could increase. In the pathological grade, the SPP1 expression was higher in LUAD samples with moderate differentiation. In addition, the overall 5-year survival rates of the SPP1 high and low expression groups were 50.574 and 59.181% [P = 0.008; hazard ratio (HR) = 0.7057; 95% CI, 0.5467–0.9109], indicating that SPP1 had an impact on overall survival for LUAD patients. The relationship between SPP1 expression and CD4+ T cell, macrophage, neutrophil, and dendritic cell infiltration was weak in LUAD. SPP1 could be considered as an independent prognostic marker in LUAD (P = 0.003; HR = 1.150; 95% CI, 1.048–1.261) by multivariate Cox regression analysis. The results of GSEA indicated that samples with high SPP1 expression were enriched in protein secretion, mTORC1 signaling, angiogenesis, and glycolysis pathway. The analysis results obtained by TCGA-LUAD data were basically consistent with the results obtained by GSE68465.ConclusionsSPP1 can not only affect the occurrence and development of LUAD but also may be an independent prognostic marker of LUAD. SPP1 is expected to be a new target for molecular targeted therapy.

BET inhibitor suppresses melanoma progression via the noncanonical NF-κB/SPP1 pathway.

Background: Bromodomain and extra-terminal domain (BET) inhibitors have shown profound efficacy against hematologic malignancies and solid tumors in preclinical studies. However, the underlying molecular mechanism in melanoma is not well understood. Here we identified secreted phosphoprotein 1 (SPP1) as a melanoma driver and a crucial target of BET inhibitors in melanoma. Methods: Bioinformatics analysis and meta-analysis were used to evaluate the SPP1 expression in normal tissues, primary melanoma, and metastatic melanoma. Real-time PCR (RT-PCR) and Western blotting were employed to quantify SPP1 expression in melanoma cells and tissues. Cell proliferation, wound healing, and Transwell assays were carried out to evaluate the effects of SPP1 and BET inhibitors in melanoma cells in vitro. A xenograft mouse model was used to investigate the effect of SPP1 and BET inhibitors on melanoma in vivo. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the regulatory mechanism of BET inhibitors on SPP1. Results: SPP1 was identified as a melanoma driver by bioinformatics analysis, and meta-analysis determined it to be a diagnostic and prognostic biomarker for melanoma. SPP1 overexpression was associated with poor melanoma prognosis, and silencing SPP1 suppressed melanoma cell proliferation, migration, and invasion. Through a pilot drug screen, we identified BET inhibitors as ideal therapeutic agents that suppressed SPP1 expression. Also, SPP1 overexpression could partially reverse the suppressive effect of BET inhibitors on melanoma. We further demonstrated that bromodomain-containing 4 (BRD4) regulated SPP1 expression. Notably, BRD4 did not bind directly to the SPP1 promoter but regulated SPP1 expression through NFKB2. Silencing of NFKB2 resembled the phenotype of BET inhibitors treatment and SPP1 silencing in melanoma. Conclusion: Our findings highlight SPP1 as an essential target of BET inhibitors and provide a novel mechanism by which BET inhibitors suppress melanoma progression via the noncanonical NF-κB/SPP1 pathway.

The non-synonymous mutation in bovine SPP1 gene influences carcass weight

Meat quality in beef cattle is controlled by genetic factors. SPP1 (secreted phosphoprotein 1) gene, coding a multifunctional cytokine with diverse biological functions, is the candidate gene influencing carcass traits. In this study, we tried to discover DNA polymorphisms associated with beef quality in bovine SPP1 gene, so that two SNPs (single nucleotide polymorphisms) in the promoter region and one SNP in the CDS (coding sequence) region were identified. Although the formers were predicted to alter SPP1 expression, they did not show any effects on the traits. On the contrary, statistical analysis revealed that g.58675C > T, a non-synonymous mutation from threonine to methionine in the conservative region, had a significant effect on carcass weight. Carcass weight of the animals with C/T allele (473.9 ± 6.0 kg) was significantly heavier than that of the C/C homozygotes (459.2 ± 2.8 kg). Because SPP1 gene functions in skeletal muscle cells as a positive regulator, the non-synonymous mutation might influence muscle development and remodeling, resulting in increased carcass weight of the C/T animals. Our results indicate that the SNP can be applied as a DNA marker for the improvement of beef cattle.

TMIC-07. SYMBIOTIC MACROPHAGE-GLIOMA CELL INTERACTIONS REVEAL SYNTHETIC LETHALITY IN PTEN NULL GLIOMA

Abstract Heterotypic interactions across diverse cell types of the tumor microenvironment (TME) can enable tumor progression and offer points for potential therapeutic intervention. Employing Glioblastoma Multiforme (GBM) as a model system, we sought to (i) understand the nature of specific oncogenic signals in glioma cells that affects immune cells, and (ii) identify immune cell factors which may support glioma growth and survival. Combined profiling and functional studies of glioma cells established that PTEN deficiency, a signature GBM alteration, specifically tracks with an immune profile dominated by tumor-associated macrophages (TAMs, a key type of immune cells in GBM which can constitute up to half of the tumor mass) via upregulation of lysyl oxidase (LOX). Mechanistically, the PTEN-SRC/AKT axis regulates YAP1, which directly upregulates LOX expression and secretion in glioma cells via binding to the LOX promoter. The secreted LOX from glioma cells functions as a potent macrophage chemoattractant to promote macrophage recruitment via activation of the beta1 integrin-PYK2 pathway in macrophages. Consequently, these infiltrating macrophages secrete SPP1 (secreted phosphoprotein 1) which sustains glioma cell survival and stimulates angiogenesis. In human and mouse PTEN-null GBM models, but not PTEN-intact GBM models, LOX inhibition markedly inhibits tumor growth and extends the survival of tumor-bearing mice by decreasing macrophage infiltration, impairing SPP1 expression and attenuating angiogenesis. In silico analysis of human GBM shows that increased YAP1-LOX and b1 integrin-SPP1 signaling positively correlates with higher macrophage density and lower overall survival. Together, the symbiotic glioma (PTEN-YAP1-LOX) and macrophage (beta1 integrin-PYK2-SPP1) interactions provide novel therapeutic targets for this intractable disease.

Identification of Secreted Phosphoprotein 1 (SPP1) as a Prognostic Factor in Lower-Grade Gliomas

Secreted phosphoprotein 1 (SPP1) is an important extracellular glycoprotein that is associated with immune regulation, tumorigenesis, and cell signaling. However, the prognostic value of SPP1 in patients with glioma has not yet been clarified, especially in lower-grade gliomas. The objective of this study is to evaluate the prognostic merit of SPP1 in lower-grade gliomas. The messenger RNA (mRNA) expression of SPP1 in about 1000 cancer cell lines was explored by using the data from the Cancer Cell Line Encyclopedia database. The Oncomine database was mined to evaluate the mRNA expression of SPP1 in lower-grade glioma, glioblastoma, and normal brain tissues. The correlation between SPP1 mRNA expression and overall survival of patients with glioma from The Cancer Genome Atlas database was analyzed. SPP1 mRNA expression of glioma was ranked as the eighth highest of all cancer cell lines in the Cancer Cell Line Encyclopedia database. The data from the Oncomine database suggested that SPP1 expression was significantly high in glioblastoma compared with normal brain tissues but was not significantly high in lower-grade glioma compared with normal brain tissue. Analysis of the RNA-Seq data from The Cancer Genome Atlas database showed that the increased SPP1 mRNA expression in lower-grade glioma was significantly associated with poor survival outcomes in patients with lower-grade glioma. Multivariate Cox regression analysis showed that SPP1 might be considered as an independent prognostic factor in lower-grade gliomas. The present study showed that SPP1 overexpression is related to worse overall survival in patients with lower-grade glioma. Moreover, SPP1 could be considered as an independent factor in lower-grade gliomas.

Genome-wide analysis reveals alcohol dehydrogenase 1C and secreted phosphoprotein 1 for prognostic biomarkers in lung adenocarcinoma.

To seek out novel promising biomarkers for predicting lung adenocarcinoma (LUAD) prognosis, we conducted this study. First, 279 upregulated and 37 downregulated differentially expressed genes were obtained from LUAD and para-carcinoma tissues by the Affymetrix GeneChip Human Transcriptome Array. Then, we randomly classified samples of LUAD data set GSE31210 as training and testing sets in a 1:1 ratio. Alcohol dehydrogenase 1C (ADH1C) and secreted phosphoprotein 1 (SPP1) were finally identified correlating with the LUAD survival through least absolute shrinkage and selection operator penalized Cox proportion hazards regression model, and applied to build a 2-gene signature related to prognosis in training set. Univariate and multivariable survival analyses suggested that overall survival (OS) and relapse-free survival (RFS) in the 2-gene signature low-risk group were better than the high-risk group. Kaplan-Meier curves proved that elevated ADH1C expression and reduced SPP1 expression were related to better OS and RFS. Besides, the SPP1 expressed higher in LUAD than para-carcinoma tissues using quantitative reverse transcription polymerase chain reaction assay. Finally, the association between the two genes and clinicopathological parameters in 80 LUAD were analyzed, it is suggested that SPP1 was relevant to epidermal growth factor receptor mutation. These findings indicated that ADH1C and SPP1 might be novel promising biomarkers for predicting LUAD prognosis.

Secreted Phosphoprotein 1 Promotes the Development of Small Cell Lung Cancer Cells by Inhibiting Autophagy and Apoptosis.

This study aimed to investigate the expression of secreted phosphoprotein 1 (SPP1) on lung cancer cells and explore its underlying mechanism on autophagy and apoptosis which effect the development of lung cancer cells. GSE19804 related to lung cancer cells was screened from Gene Expression Omnibus (GEO) database, and we screened the 47 pairs of differential expressed mRNAs in lung cancer cells and adjacent tissues using microarray analysis. The expression of the core gene SPP1 was detected by qRT-PCR and western-blot. The transfection efficiency of lung cancer cells was detected by qRT-PCR and the expression of transfected group was tested by western-blot. Cell proliferation after transfection was tested by MTT assay and plate cloning experiment. The apoptosis rate of each transfection group was detected by flow cytometry. We use western-blot to test protein expression of autophagy-related proteins Beclin-1, LC3-I, LC3-II and p62 of each transfected group. Through analysis of GSE19804,the heat map showed SPP1 was the highest expressed in tumor tissues. qRT-PCR and western-blot detected SPP1 expression in lung cancer tissues was higher than that in normal adjacent tissues and was significantly increased in lung cancer cell lines. After transfection with pcDNA3.1-SPP1 (p-SPP1 group), siRNA1-SPP1 (siRNA1 group) and siRNA2-SPP1 (siRNA2 group), showed different expression of SPP1. Up-regulation of SPP1 enhanced cell viability and promoted tumor cell proliferation, while knockdown of SPP1 inhibited tumor cell proliferation. From the results of apoptosis rate, SPP1 inhibited the tumor cell apoptosis. However, in normal lung cell, SPP1 had no effect on cell proliferation and apoptosis. And to test autophagy-related proteins, we found that overexpression of SPP1 inhibited autophagy. High expression of SPP1 inhibited autophagy and apoptosis to promote the development of small cell lung cancer cells.

Identification of SPP1 as a promising biomarker to predict clinical outcome of lung adenocarcinoma individuals

We identified and investigated the prognostic value of secreted phosphoprotein 1 (SPP1) in lung adenocarcinoma (LUAD) patients and evaluate the relationship between the SPP1 expression level and clinical characteristics. First, SPP1 was identified through four LUAD datasets in GEO database and validated by the TCGA database. Then a total of 149 lung adenocarcinoma patients were included, as well as their clinicopathological characteristics collected in the study. The expression levels of SPP1 and adjacent normal tissues were assessed by immunohistochemistry. The association between SPP1 and its prognostic value was systematically evaluated. Results showed high SPP1 level predicted poorer LUAD survival by univariate Cox analysis (P = 0.017), and the multivariate result was also significant (P = 0.021). Subgroup analysis suggested the prognostic value of SPP1 was significant in stage T1 (log rank P = 0.011), stage N0 (log rank P = 0.046) and stage N1 (log rank P = 0.009) patients. After including these markers into a nomogram, the Harrell's C-index of the nomogram was 0.694. In summary, our findings suggest that SPP1 is an independent biomarker with prognostic significance in LUAD.

Stress signaling in breast cancer cells induces matrix components that promote chemoresistant metastasis

Metastatic progression remains a major burden for cancer patients and is associated with eventual resistance to prevailing therapies such as chemotherapy. Here, we reveal how chemotherapy induces an extracellular matrix (ECM), wound healing, and stem cell network in cancer cells via the c‐Jun N‐terminal kinase (JNK) pathway, leading to reduced therapeutic efficacy. We find that elevated JNK activity in cancer cells is linked to poor clinical outcome in breast cancer patients and is critical for tumor initiation and metastasis in xenograft mouse models of breast cancer. We show that JNK signaling enhances expression of the ECM and stem cell niche components osteopontin, also called secreted phosphoprotein 1 (SPP1), and tenascin C (TNC), that promote lung metastasis. We demonstrate that both SPP1 and TNC are direct targets of the c‐Jun transcription factor. Exposure to multiple chemotherapies further exploits this JNK‐mediated axis to confer treatment resistance. Importantly, JNK inhibition or disruption of SPP1 or TNC expression sensitizes experimental mammary tumors and metastases to chemotherapy, thus providing insights to consider for future treatment strategies against metastatic breast cancer.

Regulator of G-protein signaling 5 enhances portal vein invasion in hepatocellular carcinoma.

Portal vein invasion (PVI) is a major prognostic factor in hepatocellular carcinoma (HCC). The aim of the present study was to identify molecules that regulate PVI. Sections of cancerous tissue, paired noncancerous tissue and the PVI area were collected from 3 frozen HCC sections, using laser microdissection. The present study focused on 3 upregulated molecules, integrin β3 (ITGB3), secreted phosphoprotein 1 (SPP1) and regulator of G-protein signaling 5 (RGS5), and 2 molecules that were downregulated in PVI tissue compared with cancer tissue, metallothionein 1G (MT1G) and metallothionein 1H (MT1H), as determined by cDNA microarray analysis. Reverse transcription-quantitative polymerase chain reaction analysis of 32 HCC cases revealed that RGS5 mRNA levels were significantly increased and MT1 G and MT1H mRNA levels were significantly decreased in cancerous tissue compared with noncancerous tissue. However, there was no significant difference in ITGB3 and SPP1 expression. There were no significant differences between the expression of these molecules and any clinicopathologic factors, including PVI. Immunohistochemical staining for RGS5 in 60 HCC cases demonstrated that RGS5 protein levels were higher in cancerous tissue compared with paired noncancerous tissue in 63.3% of HCC cases. Furthermore, high expression of RGS5 in cancerous tissue was significantly associated with PVI and tended to be associated with intrahepatic metastasis. Confluent multinodular type was significantly more frequent in cases with high expression of RGS5 in the cancerous tissue. Therefore, RGS5 may be a useful prognostic biomarker as well as a potential target of molecular therapy to treat HCC.

Upregulation of PD-L1 by SPP1 mediates macrophage polarization and facilitates immune escape in lung adenocarcinoma

Tumor-associated macrophages (TAMs) polarization represents a key regulatory process of tumor progression. However, the underlying mechanisms are unclear. This study aimed to investigate the relationship between secreted phosphoprotein 1 (SPP1) and TAMs in lung adenocarcinoma cells. THP-1 monocytes were differentiated into macrophages using PMA. PMA-treated THP-1 cells were co-cultured with human A549 cells culture supernatant. SPP1 expression in TAMs isolated from lung adenocarcinoma tissues and PMA-treated THP-1 cells were measured. Macrophage polarization was identified by flow cytometric analysis. Cell migration and apoptosis were assessed by Transwell migration assays and flow cytometric analysis, respectively. SPP1 is highly expressed in tumor tissues and TAMs isolated from patients with an advanced TNM stage, and also in PMA-treated THP-1 cells. Co-culture with A549 cells strongly induced SPP-1 expression as well as M2 polarization of THP-1 cells, but it had little effect on short hairpin SPP1 (shSPP1)-transfected THP-1 cells. Interestingly, programmed death ligand 1 (PD-L1), a critical regulator of M2 polarization, was downregulated in SPP1 knockdown THP-1 cells. Inhibition of PD-L1 induced a greater decline of the M2 markers IL-10 and Arg-1 but an increase in the M1 markers IL-12 and TNF-α. In addition, SPP1 knockdown in THP-1 cells can mitigate migration but promote apoptosis of A549 cells, and PD-L1 inhibition can further enhance this effect. THP-1 cells co-cultured with A549 cells attenuated CD4+ T-cell activation, whereas SPP1 inhibition restored T-cell activation. These results highlight the importance of SPP1 in mediating macrophage polarization and lung cancer evasion, suggesting a potential therapeutic target for lung cancer.

NR6A1 couples with cAMP response element binding protein and regulates vascular smooth muscle cell migration

Vascular smooth muscle cell (VSMC) migration is implicated in atherosclerosis and restenosis. Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is involved in regulating embryonic stem cell differentiation, reproduction, neuronal differentiation. Functional cooperation between cAMP response element modulator tau (CREMtau) and NR6A1 can direct gene expression in cells. cAMP response element binding protein (CREB) plays a key role in VSMC migration. In this study, we sought to determine whether CREB involved in NR6A1-modulated VSMC migration. VSMCs treated with platelet-derived growth factor-BB (PDGF-BB) displayed reduced mRNA and protein levels of NR6A1. Adenovirus-mediated expression of NR6A1 (Ad-NR6A1) could inhibit PDGF-BB- and serum-induced VSMC migration. The mRNA and protein expressions of secreted phosphoprotein 1 (SPP1) were down-regulated by NR6A1 overexpression. SPP1 promoter reporter activity was repressed by NR6A1. NR6A1 was found to physically couple with nuclear actin and the large subunit of RNA polymerase II. Furthermore, we showed that CREB interacted with NR6A1 in VSMCs. NR6A1 overexpression repressed cAMP response element (CRE) activity. ChIP assay revealed that NR6A1 bind to SPP1 promoter. Luciferase reporter assay showed that NR6A1 regulated SPP1 promoter activity via a putative CRE site. Adenovirus mediated local NR6A1 gene transfer attenuated stenosis after balloon-induced arterial injury in Sprague–Dawley rats. Taken together, this study provided experimental evidence that NR6A1 modulated SPP1 expression via its binding with CREB protein in VSMCs. We also revealed a NR6A1-CREB-SPP1 axis that serves as a regulatory mechanism for atherosclerosis and restenosis.