No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Lipocalin 2 as A Potential Diagnostic and/or Prognostic Biomarker in Prostate, Lung and Liver Cancer
Abstract
Lipocalin 2 (LCN2) is a 25 kDa secreted protein, initially purified from neutrophil granules, and mainly expressed in immune cells, hepatocytes, renal cells, prostate, cells of the respiratory tract and cardiomyocytes. LCN2 belongs to the family of lipocalins known for their ability to traffic small hydrophobic molecules such as lipids and retinoids. Due to its ability to sequester iron-containing bacterial siderophores, LCN2 plays an essential part in the innate immunity as well as in the regulation of the cellular iron metabolism. Altered LCN2 expression occurs in di- verse pathological conditions, including kidney disease, obesity, steatohepatitis, inflammation and malignant transformation. In recent years, LCN2 gained attention as a potential biomarker in cancer as it is protease resistant and thus easily detectable in blood, urine, tissue and other body fluids. In line, numerous peer-reviewed reports established the LCN2 overexpression in cancers of diverse histological background. Apparently, LCN2 has contradictory roles in dif- ferent types of cancers. While it facilitates tumorigenesis by promoting survival, growth, inva- sion and metastasis, other studies report a negative correlation of LCN2 and disease outcome. Particularly, LCN2 suppression promoted cell proliferation, migration, invasion as well as the switch from epithelial to mesenchymal state. The underlying molecular mechanisms of the complex and ambiguous role of LCN2 in malignant tumor development and progression are not completely clarified yet. The following review focuses on major findings of LCN2 as a po- tential diagnostic and/or prognostic biomarker in prostate, lung and liver cancer, representing worldwide three of the cancers with the highest estimated incidence, mortality, and prevalence. In addition, we will highlight the progress of knowledge in understanding molecular pathways and regulation processes of LCN2 in those cancer types.
... LCN2 is a 25-kDa secreted glycoprotein with various physiological and pathophysiological functions [18]. Furthermore, LCN2 has been discussed as a prognostic and/or diagnostic marker in various types of cancer including PCa [19,20]. Nevertheless, the exact mechanism on how LCN2 is connected to metastatic spread still remains unclear. ...
... Previously, we reviewed LCN2 studies performed in prostate, lung and liver cancer highlighting the contradictory role of LCN2 in those cancer types [19]. Human PC-3 cells are suitable as a metastatic model to investigate PCa progression [42,45], whereas A549 carcinoma cells are a common model to study lung cancer [46]. ...
... LCN2 is involved in various physiological and pathophysiological cellular processes. It is discussed as a prognostic and/or diagnostic marker in different types of cancer [19]. Nevertheless, there are only limited studies investigating regulatory aspects of LCN2 expression in PCa [21,22,36,41,51,62]. ...
Prostate cancer (PCa) is one of the most common and deadly cancers in men worldwide. The surrounding tumor microenvironment (TME) is important in tumor progression, as cytokines and soluble mediators including tumor necrosis factor (TNF-α) or lipocalin-2 (LCN2) can influence tumor growth and formation of metastasis. The exact mechanisms on how these pleiotropic factors affect PCa are still unknown. In this study, we showed for the first time that LCN2 mRNA and protein expression are strongly inducible by TNF-α in the highly metastatic human PCa cell line PC-3. In addition, we observed higher levels of secreted LCN2 in cell culture medium of TNF-α-treated PC-3 cells. We found that different signaling pathways such as p38, NF-κB or JNK were activated shortly after TNF-α treatment. Moreover, the mRNA levels of IL-1β and IL-8 were also significantly increased after 24 h stimulation. Mechanistically, the NF-κB pathway and the JNK signaling axis are directly responsible for LCN2 upregulation. This was shown by the fact that pretreatment with the JNK inhibitors SP600125 or JNK-IN-8 strongly downregulated phosphorylation of c-Jun protein and markedly reduced TNF-α-mediated LCN2 upregulation in PC-3 cells. Likewise, the NF-κB inhibitor QNZ was able to repress TNF-α-induced LCN2 expression in PC-3 cells. Taking into consideration that LCN2 has been described as a tumor promoting factor in PCa, our results indicate that JNK regulates LCN2 expression and unmasks the JNK signaling axis as a possible therapeutic target for patients with PCa.
... LCN2 has recently attracted the attention to its role in prognosis and diagnosis in various cancers, including colorectal and breast cancer [16,17]. We recently reviewed the potential roles of LCN2 in diagnosis and prognosis in prostate, pulmonary and hepatic cancer [18]. We came to the conclusion that LCN2 has widely heterogeneous roles in different cancer types, affecting frequently in a contradictory manner the cell proliferation, migration, invasion and metastasis pathways, leaving the presence of LCN2 in the tumor environment under the need of vast investigation [18]. ...
... We recently reviewed the potential roles of LCN2 in diagnosis and prognosis in prostate, pulmonary and hepatic cancer [18]. We came to the conclusion that LCN2 has widely heterogeneous roles in different cancer types, affecting frequently in a contradictory manner the cell proliferation, migration, invasion and metastasis pathways, leaving the presence of LCN2 in the tumor environment under the need of vast investigation [18]. ...
... Specifically in HCC, LCN2 expression has been described in several in vivo and in vitro studies, where it is always higher expressed in the tissue as well as in the serum of HCC patients compared to healthy subjects [18]. LCN2 expression inhibits proliferation, invasion and metastasis, with the ability to reverse the epithelial to mesenchymal transition process, suggesting LCN2 as a potential metastasis suppressor and a therapeutic target against HCC [19]. ...
Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly cancers worldwide. Therefore, current global research focuses on molecular tools for early diagnosis of HCC, which can lead to effective treatment at an early stage. Perilipin 5 (PLIN5) has been studied as one of the main proteins of the perilipin family, whose role is to maintain lipid homeostasis by inhibiting lipolysis. In this study, we show for the first time that PLIN5 is strongly expressed in tumors of human patients with HCC as well as in mouse livers, in which HCC was genetically or experimentally induced by treatment with the genotoxic agent diethylnitrosamine. Moreover, the secreted acute phase glycoprotein Lipocalin 2 (LCN2) established as a biomarker of acute kidney injury, is also proven to indicate liver injury with upregulated expression in numerous cases of hepatic damage, including steatohepatitis. LCN2 has been studied in various cancers, and it has been assigned roles in multiple cellular processes such as the suppression of the invasion of HCC cells and their metastatic abilities. The presence of this protein in blood and urine, in combination with the presence of α -Fetoprotein (AFP), is hypothesized to serve as a biomarker of early stages of HCC. In the current study, we show in humans and mice that LCN2 is secreted into the serum from liver cancer tissue. We also show that AFP-positive hepatocytes represent the main source for the massive expression of LCN2 in tumoral tissue. Thus, the strong presence of PLIN5 and LCN2 in HCC and understanding their roles could establish them as markers for diagnosis or as treatment targets against HCC
... Knockdown of LCN2 is known to affect tumorigenic markers and the EMT process in various cancers [12,17]. Our results show decreased protein expression of fibronectin, connexin 43 (Cx43) and nuclear factor kappa inhibitor zeta (IκBζ) when LCN2 was transiently downregulated with 35 nM siLCN2 in PC-3 cells ( Figure 2C). ...
... Presently, simple somatic mutations and copy number variations of LCN2 are more frequent described in other malignancies than PCa (Table S3). Although there is already discussion whether LCN2 can be used as a reliable biomarker [17,19], there is still a lack of understanding on its exact function in tumor progression, especially in PCa. ...
The transporter protein lipocalin-2 (LCN2) also termed neutrophil-gelatinase-associated lipocalin (NGAL) has pleiotropic effects in tumorigenesis in various cancers. Since the precise role of LCN2 in prostate cancer (PCa) is poorly understood, we aimed to elucidate its functions in PCa in vitro. For this purpose, LCN2 was transiently suppressed or permanently depleted in human PC-3 cells using siRNA or CRISPR/Cas9-mediated knockout. Effects of LCN2 suppression on expression of different tumorigenic markers were investigated by Western blot analysis and RT-qPCR. LCN2 knockout cells were analyzed for cellular changes and their ability to cope endoplasmic stress compared to parenteral PC-3 cells. Reduced LCN2 was accompanied by decreased expression of IL-1β and Cx43. In PC-3 cells, LCN2 deficiency leads to reduced proliferation, diminished expression of pro-inflammatory cytokines, lower adhesion, and disrupted F-actin distribution. In addition, IL-1β expression strongly correlated with LCN2 levels. LCN2 knockout cells showed enhanced and sustained activation of unfolded protein response proteins when treated with tunicamycin or cultured under glucose deprivation. Interestingly, an inverse correlation between phosphorylation of eukaryotic initiation factor 2 α subunit (p-eIF2α) and LCN2 expression was observed suggesting that LCN2 triggers protein synthesis under stress conditions. The finding that LCN2 depletion leads to significant phenotypic and cellular changes in PC-3 cells adds LCN2 as a valuable target for the treatment of PCa.
... 35 Furthermore, LCN2 has been reported as a potential prognostic and/ or diagnostic biomarker in various cancer types, specifically liver, prostate, and lung cancers assuming the highest prevalence, mortality, and incidence worldwide. 21,36 For instance, Zhang and colleagues demonstrated that a high expression level of LCN2 and its receptor, neutrophil gelatinase-associated lipocalin receptor (NGALR), were both associated with an overall decreased survival rate in patients with hepatocellular carcinoma. In addition, a significant correlation was found between LCN2 and NGALR expression in advanced stages, vascular invasion, and tumor recurrence. ...
Background
Lipocalin-2 (LCN2) deregulation has been reported in several types of cancer and is implicated in the proliferation, migration, angiogenesis, and progression of tumors. However, its aberrant expression has been rarely studied in nasopharyngeal carcinoma (NPC). In the present study, we investigated the expression of LCN2 in NPC patients.
Methods
In this descriptive cross-sectional study, 29 NPC and 20 non-cancerous control paraffin pathology blocks were obtained from the seven-year (2011 to 2018) archive of Razi Laboratory in Rasht, Iran. LCN2 mRNA expression was evaluated through quantitative real-time PCR. In addition, immunohistochemistry was performed to evaluate LCN2 expression at the protein level. The fold change value and total immunostaining score (TIS) were applied for quantitative evaluation. The nonparametric Mann-Whitney U test and Fisher’s exact test were used through GraphPad Prism 8.3.0 software. P<0.05 was considered statistically significant.
Results
Our results revealed that LCN2 mRNA and protein levels in NPC tissues were significantly higher than control tissues (P=0.028 and P=0.002, respectively). At the protein level, 65.51% (19/29) of NPC patients were categorized as having high LCN2 expression (TIS>3) and 34.47% (10/29) as low expression (TIS≤3). While in the control group, 25% (5/20) of subjects represented a high expression of LCN2 (TIS>3), and 75% (15/20) showed no or weak expression (TIS≤3). No significant correlation was found between the overexpression of LCN2 at the protein level and the demographic features of the patients.
Conclusion
Our findings suggest that LCN2 might be considered a potential new diagnostic marker for NPC. However, this warrants further studies.
... LCN2 is a 25-kDa glycoprotein that has been linked to an increasingly broad range of immunological and metabolic functions [13][14][15]. Clinically, LCN2 belongs to the lipocalin family of proteins and is already considered an important marker for prognosis and diagnosis in acute kidney injury and various malignancies [12,[16][17][18]. This multifaceted importance derives from its structure and associated functionality. ...
Estrogen receptor alpha (ERα) is widely expressed in reproductive organs, but also in non-reproductive tissues of females and males. There is evidence that lipocalin 2 (LCN2), which has diverse immunological and metabolic functions, is regulated by ERα in adipose tissue. However, in many other tissues, the impact of ERα on LCN2 expression has not been studied yet. Therefore, we used an Esr1-deficient mouse strain and analyzed LCN2 expression in reproductive (ovary, testes) and non-reproductive tissues (kidney, spleen, liver, lung) of both sexes. Tissues collected from adult wild-type (WT) and Esr1-deficient animals were analyzed by immunohistochemistry, Western blot analysis, and RT-qPCR for Lcn2 expression. In non-reproductive tissues, only minor genotype- or sex-specific differences in LCN2 expression were detected. In contrast, significant differences in LCN2 expression were observed in reproductive tissues. Particularly, there was a strong increase in LCN2 in Esr1-deficient ovaries when compared to WTs. In summary, we found an inverse correlation between the presence of ERα and the expression of LCN2 in testes and ovaries. Our results provide an important basis to better understand LCN2 regulation in the context of hormones and in health and disease.
... Structurally, it belongs to the lipocalin protein family sharing a highly compact hydrophobic β-barrel fold adapted to bind and transport a variety of hydrophobic ligands. Altered LCN2 expression occurs under diverse pathological conditions, including kidney disease, obesity, and acute and chronic inflammatory liver disease [2][3][4]. Upon intoxication, infection, inflammation, and other forms of cellular stress, LCN2 is rapidly induced in injured hepatocytes [5]. As a characteristic of an acute phase protein, LCN2 expression is stimulated by inflammatory cytokines including IL-6, IL-1β, and TNF-α [6,7]. ...
Lipocalin 2 (LCN2) mediates key roles in innate immune responses. It has affinity for many lipophilic ligands and binds various siderophores, thereby limiting bacterial growth by iron sequestration. Furthermore, LCN2 protects against obesity and metabolic syndrome by interfering with the composition of gut microbiota. Consequently, complete or hepatocyte-specific ablation of the Lcn2 gene is associated with higher susceptibility to bacterial infections. In the present study, we comparatively profiled microbiota in fecal samples of wild type and Lcn2 null mice and show, in contrast to previous reports, that the quantity of DNA in feces of Lcn2 null mice is significantly lower than that in wild type mice (p < 0.001). By using the hypervariable V4 region of the 16S rDNA gene and Next-Generation Sequencing methods, we found a statistically significant change in 16 taxonomic units in Lcn2-/- mice, including eight gender-specific deviations. In particular, members of Clostridium, Escherichia, Helicobacter, Lactococcus, Prevotellaceae_UCG-001 and Staphylococcus appeared to expand in the intestinal tract of knockout mice. Interestingly, the proportion of Escherichia (200-fold) and Staphylococcus (10-fold) as well as the abundance of intestinal bacteria encoding the LCN2-sensitive siderphore enterobactin (entA) was significantly increased in male Lcn2 null mice (743-fold, p < 0.001). This was accompanied by significant higher immune cell infiltration in the ileum as demonstrated by increased immunoreactivity against the pan-leukocyte protein CD45, the lymphocyte transcription factor MUM-1/IRF4, and the macrophage antigen CD68/Macrosialin. In addition, we found a higher expression of mucosal mast cell proteases indicating a higher number of those innate immune cells. Finally, the ileum of Lcn2 null mice displayed a high abundance of segmented filamentous bacteria, which are intimately associated with the mucosal cell layer, provoking epithelial antimicrobial responses and affecting T-helper cell polarization.
Diabetic retinopathy is a common complication of diabetes mellitus that causes pathogenic damage to the retina. Particularly, the proliferative diabetic retinopathy (PDR) state can cause abnormal angiogenesis in the retina tissues and trigger the retina destruction in advanced stage. In the clinic, the symptoms during the initiation and progression of PDR are relatively unrecognizable. Therefore, various studies have focused on the pathogenesis of PDR. According to published literature, genetic contributions play an irreplaceable role in the initiation and progression of PDR. Although many computational methods, such as shortest path- and random walk with restart-based methods, have been applied in screening the potential pathogenic factors of PDR, advanced computational methods, which may provide essential supplements for previous ones, are still widely needed. In this study, a novel computational method was presented to infer novel PDR-associated genes. Different from previous methods, the method used in this work employed a different network algorithm, that is, the Laplacian heat diffusion algorithm. This algorithm was applied on the protein–protein interaction network reported in the STRING database. Three screening tests were performed to filter the most likely inferred genes. A total of 26 genes were accessed using the proposed method. Compared with the two previous predictions, most of the identified genes were novel, and only one gene was shared. Several inferred genes, such as CSF3, COL18A1, CXCR2, CCR1, FGF23, CXCL11, and IL13, were related to the pathogenesis of PDR.
Cancer cells exhibit an increasing iron demand associated with the tumor progression. But the mechanism of iron accumulation in the tumor microenvironment is still unclear. Tumor associated macrophages (TAMs) in the tumor microenvironment may act as extra iron source. However, evidence is still lacking in TAMs as iron donors. In the present study, we found that iron concentration was significantly increased at tumor metastatic stage, which could be attributed to up-regulated expression of lipocalin2 (Lcn2). TAMs in the microenvironment secreted Lcn2. Moreover, TAMs increased intracellular iron concentration in tumor cells via Lcn2 as transporter, which could be restored by Lcn2 antibody neutralization. In conclusion, TAMs increased intracellular iron concentration of the tumor cells via Lcn2 which acted as an iron transporter. Targeting Lcn2 secretion in TAMs to "starve cancer cells" could act as alternative option for tumor therapy.
Gene mutations play critical roles during cancer development and progression, and therefore represent targets for precision medicine. Here we recapitulated the pharmacogenomic data to delineate novel candidates for actionable mutations and therapeutic target drugs. As a proof-of-concept, we demonstrated that the loss-of-function of SULF2 by mutation (N491K) or inhibition enhanced sorafenib sensitivity in liver cancer cells and in vivo mouse models. This effect was mediated by deregulation of EGFR signaling and downstream expression of LCN2. We also report that the liver cancer patients non-responding to sorafenib treatment exhibit higher expression of SULF2 and LCN2. In conclusion, we suggest that SULF2 plays a key role in sorafenib susceptibility and resistance in liver cancer via deregulation of LCN2. Diagnostic or therapeutic targeting of SULF2 (e.g., OKN-007) and/or LCN2 can be a novel precision strategy for sorafenib treatment in cancer patients.
It has been drawn attention that secreted proteins with signal peptide from cancer cells provide new potential biomarkers of cancer. In this study, three lung adenocarcinoma cell lines and serum samples from 20 patients were used for identifying potential serologic tumor biomarker with proteomic and bioinformatics approaches. One-dimensional electrophoresis, and identified with mass spectrometry and database research were performed. We found17 secreted proteins in common, while another 17 proteins with signal peptide were identified in all three lung adenocarcinoma cell lines alone with patient samples. With matching these two groups of identified proteins, calsyntenin-1 (CLSTN1), clusterin (CLU) and neutrophil gelatinase-associated lipocalin (NGAL) were found highly secreted from both cell lines and serum with unique signal peptides. Therefore, in our study, we demonstrated that cancer cells secret specific proteins to the environment that may serve as unique markers for cancer diagnosis. To combination of proteomic study with bioinformatic prediction on signal peptides, higher expression level of CLSTN1, CLU and NGAL were found and may be new solid serologic biomarkers for patients with lung adenocarcinoma.
Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most frequently occurring types of primary liver cancer and together are among the most common incident cancers worldwide. There are a number of modifiable and nonmodifiable HCC and ICC risk factors that have been reported. A review of the existing literature the epidemiology and risk factors for HCC and ICC was performed. There are a number of major infectious, lifestyle, metabolic, and heritable risk factors for both HCC and ICC. Some of these risk factors are either potentially preventable (eg, alcohol and tobacco use) or are currently treatable (eg hepatitis infection). In most cases, the molecular pathway or mechanism by which these etiologic factors cause primary liver cancer has not been well delineated. However, in nearly all cases, it is believed that a given risk factor causes liver injury and inflammation which results in chronic liver disease. Given the rising prevalence of several common HCC and ICC risk factors in the western world, the best opportunities for improving the care of these patients are either through the prevention of modifiable risk factors that are associated with the development of chronic liver disease or the identification of at risk patients, ensuring they are appropriately screened for the development of primary liver cancer, and initiating treatment early.
Lipocalin 2 (LCN2), a member of the lipocalin superfamily, plays an important role in oncogenesis and progression in various types of cancer. However, the expression pattern and functional role of LCN2 in colorectal cancer (CRC) is still poorly understood. The purpose of this study was to investigate whether LCN2 is associated with proliferation and the epithelial-mesenchymal transition (EMT) in CRC and to elucidate the underlying signaling pathways. LCN2 was preferentially expressed in CRC cells compared to normal tissues. However, LCN2 expression was significantly lower in metastatic or advanced-stage CRC than in non-metastatic or early stage CRC. Knockdown of LCN2 using small interfering RNA (siRNA) in CRC cells expressing a high level of LCN2 induced cell proliferation and a morphological switch from an epithelial to mesenchymal state. Furthermore, down-regulation of LCN2 in CRC cells increased cell migration and invasion involved in the regulation of EMT markers. Knockdown of LCN2 also induced glucose consumption and lactate production, accompanied by an increase in energy metabolism-related genes. Taken together, our findings indicated that LCN2 negatively modulated proliferation, EMT and energy metabolism in CRC cells. Accordingly, LCN2 may be a candidate metastasis suppressor and potential therapeutic target in CRC.
Since the 1970s, the epidemic of hepatocellular carcinoma (HCC) has spread beyond the Eastern Asian predominance and has been increasing in Northern hemisphere, especially in the United States (US) and Western Europe. It occurs more commonly in males in the fourth and fifth decades of life. Among all cancers, HCC is one of the fastest growing causes of death in the US and poses a significant economic burden on healthcare. Chronic liver disease due to hepatitis B virus or hepatitis C virus and alcohol accounts for the majority of HCC cases. Incidence of nonalcoholic fatty liver disease has been on the risem and it has also been associated with the development of HCC. Its pathogenesis varies based on the underlying etiological factor although majority of cases develop in the setting of background cirrhosis. Carcinogenesis of HCC includes angiogenesis, chronic inflammation, and tumor macroenvironment and microenvironment. There is a significant role of both intrinsic genetic risk factors and extrinsic influences such as alcohol or viral infections that lead to the development of HCC. Understanding its etiopathogenesis helps select appropriate diagnostic tests and treatments.
Background: Lung cancer is the leading cause of cancer-related mortality in both men and women throughout the world. The need to detect lung cancer at an early, potentially curable stage, is essential and may reduce mortality by 20%. The aim of this study was to identify distinct proteomic profiles in bronchoalveolar fluid (BALF) and plasma that are able to discriminate individuals with benign disease from those with non-small cell lung cancer (NSCLC).
Methods: Using label-free mass spectrometry analysis of BALF during discovery-phase analysis, a significant number of proteins were found to have different abundance levels when comparing control to adenocarcinoma (AD) or squamous cell lung carcinoma (SqCC). Validation of candidate biomarkers identified in BALF was performed in a larger cohort of plasma samples by detection with enzyme-linked immunoassay.
Results: Four proteins (Cystatin-C, TIMP-1, Lipocalin-2 and HSP70/HSPA1A) were selected as a representative group from discovery phase mass spectrometry BALF analysis. Plasma levels of TIMP-1, Lipocalin-2 and Cystatin-C were found to be significantly elevated in AD and SqCC compared to control.
Conclusion: The results presented in this study indicate that BALF is an important proximal biofluid for the discovery and identification of candidate lung cancer biomarkers.
General significance: There is good correlation between the trend of protein abundance levels in BALF and that of plasma which validates this approach to develop a blood biomarker to aid lung cancer diagnosis, particularly in the era of lung cancer screening. The protein signatures identified also provide insight into the molecular mechanisms associated with lung malignancy.
Cholangiocarcinoma (CCA) is a devastating disease due to no effective treatments available. Since the non-mineral functions of vitamin D emerges, 1α,25(OH)2D3, the active form of vitamin D, has been applied in anti-cancer researches. In this study, we demonstrated that both the 1α,25(OH)2D3 analog, MART-10, and 1α,25(OH)2D3 possessed anti-growth effect on human CCA cells with MART-10 much more potent than 1α,25(OH)2D3. The growth inhibition of both drugs were mediated by induction of G0/G1 cell cycle arrest through upregulation of p27 and downregulation of CDK4, CDK6, and cyclin D3. Human neutrophil gelatinase associated lipocalin (NGAL) was found to be involved in 1α,25(OH)2D3 and MART-10 meditated growth inhibition for CCA as knockdown of NGAL decreased Ki-67 expression in SNU308 cells and rendered SNU308 cells less responsive to 1α,25(OH)2D3 and MART-10 treatment. Vitamin D receptor (VDR) knockdown partly abolished MART-10-induced inhibition of NGAL and cell growth in SNU308 cells. The xenograft animal study demonstrated MART-10 could effectively repressed CCA growth in vivo without inducing obvious side effects. The IHC examination of human CCA specimen for VDR revealed that higher VDR expression was linked with better prognosis. Collectively, our results suggest that MART-10 could be a promising regimen for CCA treatment.
This study was conducted to evaluate the safety and effectiveness of localized lung resection combined with neoadjuvant chemotherapy in the treatment of stage I–II non-small cell lung cancer (NSCLC). A total of 88 patients, who were admitted to our hospital for first diagnosis and treatment, were selected. The patients were divided into control group (n=40 cases) and observation group (n=48 cases) according to the last digit of the admission number. The control group was treated with minimally invasive localized lung resection by thoracoscope. The observation group underwent the same procedure combined with two cycles of systemic neoadjuvant chemotherapy before the surgery was adopted in the observation group. The effects of both treatments were compared. The operation time, intraoperative blood loss and postoperative drainage volume of observation group were significantly lower than those of the control group (P<0.05). The surgical resection rate and margin negative rate of observation group were higher than those of control group, while the occurrence rate of complications was lower than that of control group; results were statistically significant (P<0.05). The serum neutrophil gelatinase associated lipocalin (lipocalin-2/NGAL), matrix metalloproteinase-9 (MMP-9) and carcinoembryonic antigen (CEA) levels of two groups after the treatment were lower than those before; however, levels in the observation group exhibited a distinct decrease. The difference has statistical significance (P<0.05). The follow-up time of two groups was 3–38 months and the median time was 20.5 months. The tumor survival period of observation group was not prolonged, however, the survival rate and quality rate of life were enhanced; the difference has statistical significance (P<0.05). Localized lung resection combined with neoadjuvant chemotherapy can effectively improve the surgical effect of stage I–II NSCLC, prolong the survival period, enhance the survival rate, decrease the occurrence rate of complications and reduce the tumor related factors lipocalin-2, MMP-9 and CEA levels.
Background:
Some studies have reported differentially altered neutrophil gelatinase-associated lipocalin (NGAL) levels in several malignancies. We evaluated NGAL measured in plasma or urine as both prognostic and diagnostic marker for different types of human tumors.
Methods:
We performed systematic electronic searches in Medline, Embase and CRDTAS. Studies were included if they evaluated NGAL as a prognostic or diagnostic marker for human cancers. The selection of the studies, screening of the full texts and data extraction were conducted independently by 2 authors. We used the random-effects model for the meta-analyses. A methodological assessment was completed.
Results:
We included 35 studies dedicated to colorectal, pancreas, breast, thyroid, gastric, kidney, endometrial, brain, liver, lung, esophageal, oral and ovarian cancers. Our meta-analyses showed that, in patients with colorectal and breast cancer, positive NGAL expression was associated with a decrease of disease-free survival (hazard ratio [HR] = 2.27, 95% confidence interval [95% CI], 1.54-3.36; HR = 1.78, 95% CI, 1.33-2.38, respectively). NGAL was a negative prognostic marker of overall survival in colorectal (HR = 2.37, 95% CI, 1.68-3.34) and endometrial (HR = 4.38, 95% CI, 1.9-10.12) cancers. Discriminative power of NGAL between cancer patients and control was moderate in colorectal cancer (area under the curve [AUC] = 0.6; pooled sensitivity 0.56; pooled specificity 0.72), acceptable in pancreatic cancer (AUC = 0.8; pooled sensitivity 0.6; pooled specificity 0.8) and good in thyroid cancer (AUC = 0.9; pooled sensitivity 0.85; pooled specificity 0.96).
Conclusions:
NGAL determination in plasma and urine could be useful in the prognosis of colorectal and breast cancer, but its prognostic accuracy remains uncertain for other human tumors.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Due to the high incidence of post-operative recurrence after current treatments, the identification of new and more effective drugs is required. In previous years, new targetable genes/pathways involved in HCC pathogenesis have been discovered through the help of high-throughput sequencing technologies. Mutations in TP53 and β-catenin genes are the most frequent aberrations in HCC. However, approaches able to reverse the effect of these mutations might be unpredictable. In fact, if the reactivation of proteins, such as p53 in tumours, holds great promise as anticancer therapy, there are studies arguing that chronic activation of these types of molecules may be deleterious. Thus, recently the efforts on potential targets have focused on actionable mutations, such as those occurring in the gene encoding for focal adhesion kinase (FAK). This tyrosine kinase, localized to cellular focal contacts, is over-expressed in a variety of human tumours, including HCC. Moreover, several lines of evidence demonstrated that FAK depletion or inhibition impair in vitro and in vivo HCC growth and metastasis. Here, we provide an overview of FAK expression and activity in the context of tumour biology, discussing the current evidence of its connection with HCC development and progression.
Background:
Castration-resistant prostate cancer (CRPC) is the lethal phenotype of prostate cancer. Lipocalin 2 (LCN2) is aberrantly expressed in many cancers including primary prostate cancer (PCa), but its role in CRPC has not been reported.
Results:
LCN2 expression was upregulated in human primary PCa and CRPC tissues. Overexpression of LCN2 promoted C4-2B and 22RV1 cell proliferation while knockdown of LCN2 markedly inhibited C4-2B and 22RV1 cell growth. LCN2 overexpression led to increased AR downstream gene SLC45A3 without upregulating AR expression. In the xenograft model, overexpression of LCN2 significantly promoted tumor growth.
Methods:
LCN2 expression was detected in primary PCa and CRPC tissues and cell lines C4-2B and 22RV1 using immunohistochemistry and western blotting, respectively. Serum LCN2 level was detected vi ELISA. Lentiviruses-mediated over-expression of LCN2 and LCN2 knockdown were performed in CRPC cell lines. Expressions of androgen receptor(AR) downstream genes was examined in cell lines, in CRPC tissues, and in animal models.
Conclusion:
LCN2 could facilitate cell proliferation of CRPC via AR transcriptional activity. LCN2 could be a novel target in CRPC.
A 25-kDa protein was found to be associated with purified human neutrophil gelatinase. Polyclonal antibodies raised against gelatinase not only recognized gelatinase but also this 25-kDa protein. Specific antibodies against the 25-kDa protein were obtained by affinity purification of the gelatinase antibodies. Immunoblotting and immunoprecipitation studies demonstrated the 135-kDa form of gelatinase to be a complex of 92-kDa gelatinase and the 25-kDa protein, and the 220-kDa form was demonstrated to be a homodimer of the 92-kDa protein, thus explaining the 220-, 135-, and 92-kDa forms characteristic of neutrophil gelatinase. The 25-kDa protein was purified to apparent homogeneity from exocytosed material from phorbol myristate acetate-stimulated neutrophils. The primary structure of the 25-kDa protein was determined as a 178-residue protein. It was susceptible to treatment with N-glycanase, and one N-glycosylation site was identified. The sequence did not match any known human protein, but showed a high degree of similarity with the deduced sequences of rat alpha2-microglobulin-related protein and the mouse protein 24p3. It is thus a new member of the lipocalin family. The function of the 25-kDa protein, named neutrophil gelatinase-associated lipocalin (NGAL), remains to be determined.
Cervical carcinoma is the third-most common cause of cancer-related deaths in women worldwide. However, the molecular mechanisms underlying the metastasis of cervical cancer are still unclear. Oligonucleotide microarrays coupled with bioinformatics analysis show that cytoskeletal remodeling and epithelial-to- mesenchymal transition (EMT) are significant pathways in clinical specimens of cervical cancer. In accord with clinical observations demonstrating ectopic expression of lipocalin 2 (LCN2), an oncogenic protein associated with EMT, in malignant tumors, was significantly upregulated in cervical cancer and correlated with lymph node metastasis. Overexpression of LCN2 enhanced tumor cell migration and invasion both in vitro and in vivo. Conversely, knockdown or neutralization of LCN2 reduced tumor cell migration and invasion. LCN2-induced migration was stimulated by activation of the EMT-associated proteins, Snail, Twist, N-cadherin, fibronectin, and MMP-9. Our findings collectively support a potential role of LCN2 in cancer cell invasion through the EMT pathway and suggest that LCN2 could be effectively utilized as a lymph node metastasis marker in cervical cancer.
Lipocalin 2 (LCN2), a multifunctional secretory protein known as neutrophil gelatinase-associated lipocalin (NGAL), is expressed
in a variety of cancers. However, little is known about the biological functions of NGAL in the development of lung adenocarcinoma.
In the present study, we primarily found that NGAL expression was up-regulated in human lung adenocarcinoma tissues. Additionally,
depletion of NGAL expression decreased the ability of cell proliferation and induced cell apoptosis. Furthermore, with the
addition of N-acetylcysteine, a scavenger of reactive oxygen species (ROS), it was found that NGAL depletion was sufficient to cause apoptosis
of lung adenocarcinoma cells by generating ROS through the inhibition of the nuclear factor E2-related factor 2/heme oxygenase-1
anti-oxidant pathway. Finally, the effect of NGAL down-regulation on the growth of human lung adenocarcinoma was determined
in BALB/c nude mice. These findings demonstrate that NGAL may be a potential therapy target for patients with lung adenocarcinoma.
We validated a single-stranded, DNA aptamer-based, diagnostic method capable of detecting Lipocalin-2 (LCN2), a biomarker from clinically relevant hepatocellular carcinoma (HCC) patient serum, in the sandwich assay format. Nine aptamers (LCN2_apta1 to LCN2_apta9) for LCN2 were screened with SELEX processes, and a sandwich pair (LCN2_apta2 and LCN2_apta4) was finally chosen using surface plasmon resonance (SPR) and dot blotting analysis. The result of the proposed aptamer sandwich construction shows that LCN2 was sensitively detected in the concentration range of 2.5-500 ng mL(-1) with a limit of detection of 0.6 ng mL(-1). Quantitative measurement tests in HCC patients were run on straight serum and were compared with the performance of the conventional antibody-based ELISA kit. The aptamer sandwich assay demonstrated an excellent dynamic range for LCN2 at clinically relevant serum levels, covering sub-nanogram per mL concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. It consists of functionalization, hybridization and signal read-out, and no dilution is required. The results of the study demonstrate the capability of the aptamer sandwich assay platform for diagnosing HCC and its potential applicability to the point-of-care testing (POCT) system.
The thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), regulates cell growth, development and differentiation via interactions with thyroid hormone receptors (TR), but the mechanisms underlying T3-mediated modulation of cancer progression are currently unclear. Lipocalin 2 (LCN2), a tumor-associated protein, is overexpressed in a variety of cancer types. Oligonucleotide microarray, coupled with proteomic analysis, has revealed that LCN2 is positively regulated by T3/TR. However, the physiological role and pathway of T3-mediated regulation of LCN2 in hepatocellular carcinogenesis remain to be characterized. Upregulation of LCN2 after T3 stimulation was observed in a time- and dose-dependent manner. Additionally, TRE on the LCN2 promoter was identified at positions -1444/-1427. Overexpression of LCN2 enhanced tumor cell migration and invasion, and conversely, its knockdown suppressed migration and invasion, both in vitro and in vivo. LCN2-induced migration occurred through activation of the Met/FAK cascade. LCN2 was overexpressed in clinical hepatocellular carcinoma (HCC) patients, compared with normal subjects, and positively correlated with TRα levels. Both TRα and LCN2 showed similar expression patterns in relation to survival rate, tumor grade, tumor stage and vascular invasion. Our findings collectively support a potential role of T3/TR in cancer progression through regulation of LCN2 via the Met/FAK cascade. LCN2 may thus be effectively utilized as a novel marker and therapeutic target in HCC.
Hepatocellular carcinoma is one of the most prevalent life-threatening human cancers with etiological factors chronic viral hepatitis B and C infections. Tumor cell dispersion relies on the loss of homotypic cell-cell adhesion. Invasion through basement membrane and interstitial extracellular matrix is another key event for metastatic progression, which requires the action of a series of proteolytic enzymes named matrix metalloproteinases, secreted by tumor cells that enhance tumor invasiveness and metastasis. TIMPs are dominant inhibitors of MMPs and able to control MMP-mediated ECM breakdown by binding active forms of MMPs. Lipocalin-2 is known as neutrophil gelatinase associated lipocalin promotematrix degradation and tumor progression. Aim: To evaluate the importance of lipocalin for diagnosis HCC in Egyptian chronic liver disease patients. Subjects and Methods: 50 patients and 25 controls. (G-1) 25 HCC on top of hepatitis C. (G-2) 25 hepatitis C. The following done: schistosoma antibodies, ASAM, LKM-1, ANA AKA and CBC. Hepatitis B surface antigen, Hepatitis C antibodies AFP, Cupper and zinc, Matrix metaloprotinase-9, TIMP-1 and Neutrophil gelatinase-associated lipocalin. Results: Median value of MMP-9 level in G-I (206 µg/L) is significantly higher than G-2 (100 µg/L) and G 3 (49 µg/L) p < 0.001. TIMP-9 median value G-1 (48 µg/L) is significantly lower than G-2 (54 µg/L) and G-3 (113 µg/L) p < 0.001. Lipocalin-2 median levels are significantly higher in G-I (389 ng/mL) than G-2 (166 ng/ml) versus G-3 (60 ng/mL) p < 0.001. Lipocalin-2 associated with increasing lobular inflammation, ballooning & fibrosis with MMP-9 has an important role in pathogenesis of liver cirrhosis and HCC. Conclusion: We elucidated predictive value for MMPs, TIMPs, and progression metastasis of hepatocellular carcinoma. Liver cell impairment alters metabolism of trace metals as zinc and copper, with possible relationship of these changes to pathogenesis of chronic liver disease. Lipocalin-2 can be used as a future diagnostic marker with better sensitivity and specificity than MMP-9 for the progression of hepatocelluar carcinoma.
Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer. Vitamin D supplementation is getting popular due to its anti-tumor functions after conversion to its active form, 1α,25(OH)2D. Here, we show that dietary supplementation with 6 IU/g of vitamin D greatly suppressed ICC initiation and progression without apparent toxicity in a chemically induced rat model. Microarray analysis of rat ICC tissues showed vitamin D supplementation modulated the expressions of several unique genes, including lipocalin 2 (Lcn2), confirmed by RT-qPCR and immunohistochemical (IHC) staining. Further, 53 of 80 human ICC specimens (66%) exhibited high LCN2 expression and LCN2 knockdown in SNU308 cells decreased cell growth and migration, suggesting LCN2 be an oncogene in human ICC. As human ICC SNU1079 cells were treated by 1α,25(OH)2D3, LCN2 expression and cell proliferation were attenuated. The downregulation of LCN2 expression was blunted when vitamin D receptor (VDR) was knocked down, implicating that the in vivo Lcn2 downregulation is a direct consequence of vitamin D supplementation
Our results support the prevailing concept that vitamin D status is negatively associated with cancer incidence and mortality and suggest LCN2 may be a potential target against ICC. Further studies of application of vitamin D or its analogs against ICC are warranted.
Cancer remains one of the major cause of death in the Western world. Although, it has been demonstrated that new therapies can improve the outcome of cancer patients, still many patients relapse after treatment. Therefore, there is a need to identify novel factors involved in cancer development and/or progression. Recently, neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as a key player in different cancer types. Its oncogenic effect may be related to the complex NGAL/MMP-9. In the present study, NGAL was analyzed at both transcript and protein levels in different cancer types by analysing 38 public available microarray datasets and the Human Protein Atlas tool.
NGAL transcripts were significantly higher in the majority of solid tumors compared to the relative normal tissues for every dataset analyzed. Furthermore, concordance of NGAL at both mRNA and protein levels was observed for 6 cancer types including bladder, colorectal, liver, lung, ovarian, and pancreatic. All metastatic tumors showed a decrease of NGAL expression when compared to matched primary lesions.
According to these results, NGAL is a candidate marker for tumor growth in a fraction of solid tumors. Further investigations are required to elucidate the function of NGAL in tumor development and metastatic processes.
Though prostate cancer (PCa) has slow progression, the hormone refractory (HRCP) and metastatic entities are substantially lethal and lack effective treatments. Transcription factor Slug is critical in regulating metastases of various tumors including PCa. Here we studied targeted therapy against Slug using combination of 3 drugs targeting 3 pathways respectively converging via Slug and further regulating PCa metastasis. Using in vitro assays we confirmed that Slug up-regulation incurred inhibition of E-cadherin that was anti-metastatic, and inhibited Bim-regulated cell apoptosis in PCa. Upstream PTEN/Akt, mTOR, Erk, and AR/Hsp90 pathways were responsible for Slug up-regulation and each of these could be targeted by rapamycin, CI-1040, and 17-AAG respectively. In 4 PCa cell lines with different traits in terms of PTEN loss and androgen sensitivity we tested the efficacy of mono- and combined therapy with the drugs. We found that metastatic capacity of the cells was maximally inhibited only when all 3 drugs were combined, due to the crosstalk between the pathways. 17-AAG decreases Slug expression via blockade of HSP90-dependent AR stability. Combination of rapamycin and CI-1040 diminishes invasiveness more potently in PCa cells that are androgen insensitive and with PTEN loss. Slug inhibited Bim-mediated apoptosis that could be rescued by mTOR/Erk/HSP90 inhibitors. Using mouse models for circulating PCa DNA quantification, we found that combination of mTOR/Erk/HSP90 inhibitors reduced circulating PCa cells in vivo significantly more potently than combination of 2 or monotherapy. Conclusively, combination of mTOR/Erk/Hsp90 inhibits metastatic capacity of prostate cancer via Slug inhibition.
The EGFR tyrosine kinase inhibitors (TKIs) demonstrate efficacy in NSCLC patients whose tumors harbor activating EGFR mutations. However, patients who initially respond to EGFR TKI treatment invariably develop resistance to the drugs. Known mechanisms account for approximately 70% of native and acquired EGFR TKI resistance. In the current study we investigated a novel mechanism of NSCLC resistance to erlotinib. Experimental Design: The mechanisms of acquired erlotinib resistance were evaluated by microarray analysis in thirteen NSCLC cell lines and in vivo in mice. Correlations between plasma neutrophil gelatinase associated lipocalin (NGAL) levels, erlotinib response and the EGFR mutational status were assessed in advanced stage NSCLC patients treated with erlotinib.
In 5 of 13 NSCLC cell lines NGAL was significantly upregulated. NGAL knockdown in erlotinib-resistant cells increased erlotinib sensitivity in vitro and in vivo. NGAL overexpression in erlotinib-sensitive cells augmented apoptosis resistance. This was mediated by NGAL-dependent modulation of the pro-apoptotic protein Bim levels. Evaluation of the plasma NGAL levels in NSCLC patients that received erlotinib revealed that patients with lower baseline NGAL demonstrated a better erlotinib response. Compared to patients with wild type EGFR, patients with activating EGFR mutations had lower plasma NGAL at baseline and weeks 4 and 8.
Our studies uncover a novel mechanism of NGAL-mediated modulation of Bim levels in NSCLC that might contribute to TKI resistance in lung cancer patients. These findings provide the rationale for the further investigations of the utility of NGAL as a potential therapeutic target or diagnostic biomarker.
HER2-positive breast cancer initially responds to trastuzumab treatment, but over time, resistance develops and rapid cancer progression occurs, for which various explanations have been proposed. Here we tested the hypothesis that induction of the unfolded protein response (UPR) could override HER2 inhibition by trastuzumab, leading to the re-activation of growth signaling and the activation of the downstream target Lipocalin 2 (LCN2). Trastuzumab significantly inhibited the basal expression of LCN2 in HER2(+) SKBr3 human breast cancer cells. The induction of the UPR completely abrogated trastuzumab-mediated LCN2 downregulation, and, in fact caused an increase in transcription and secretion of LCN2 over baseline. Reduction of the UPR using 4-phenyl butyric acid (PBA) a chemical chaperone that ameliorates ER stress, restored trastuzumab-mediated inhibition. Inhibition of the PI3K/AKT signaling pathway in trastuzumab-treated/UPR-induced SKBr3 cells partially reduced the upregulation of LCN2. These results suggest that the UPR is a possible way to override the effect of trastuzumab in HER2(+) cancer cells.
Tumor invasion is a critical step in the spread of cancer. S2R (sigma-2 receptor)/Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b(5)-related drug-binding orphan receptor essential for tumor formation and invasion. Secretory proteins drive these processes, so we screened for S2R(Pgrmc1)-dependent secreted proteins using antibody arrays. S2R(Pgrmc1) markedly regulated the expression of NGAL/LCN2 (neutrophil gelatinase-associated lipocalin/lipocalin 2), a secreted glycoprotein that binds to MMP-9 (matrix metalloproteinase 9) and protects it from degradation. S2R(Pgrmc1) knock-down blocked NGAL/LCN2 expression at the protein and RNA levels and decreased MMP9 activity. NGAL expression was required for MMP-9 activity and tumor formation. S2R(Pgrmc1) associates with EGFR and increases EGFR levels at the plasma membrane, and the EGFR inhibitors erlotinib and AG1478, as well as Akt and ERK inhibitors, suppressed the NGAL/LCN2 RNA and protein levels. NGAL is transcriptionally regulated by NFκB, and S2R(Pgrmc1) knock-down decreased the NFκB subunit p65/RelA acetylation, phosphorylation, and activation. In S2R(Pgrmc1) knock-down cells, p65 acetylation was reversed by inhibitors of histone deacetylase 1, and the inhibitors partially restored NGAL levels. Our results are consistent with a model in which S2R(Pgrmc1) increases NGAL/LCN2 levels by activating NFκB via EGFR.
Matrix metalloproteinases (MMPs) have been implicated in mechanisms of metastasis in experimental cancer models and in human malignancies. In this study, we used substrate gel electrophoresis (zymography) to determine the frequency of detection of MMPs in urine of patients with a variety of cancers. Three molecular weight classes of urinary MMPs, Mr 72,000, Mr 92,000, and high molecular weight (Mr > or = 150,000) species, were detected reproducibly and correlated with disease status. The Mr 72,000 and Mr 92,000 species were identified as MMP-2 and MMP-9, respectively, by Western blot analysis. The presence of biologically active MMP-2 (P < 0.001) or MMP-9 (P = 0.002) was an independent predictor of organ-confined cancer, and the high molecular weight species (P < 0.001) was an independent predictor of metastatic cancer. This is the first study to demonstrate that analysis of urinary MMPs may be useful in determining disease status in a variety of human cancers, both within and outside of the urinary tract.
Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production.
We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways.
Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation.
We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.
Neutrophil gelatinase-associated lipocalin (NGAL a.k.a lipocalin 2, lnc2) is a secreted protein which can form a complex with matrix metalloproteinase-9 (MMP9). This MMP9/NGAL complex has been associated with metastasis. MMP9 and NGAL are detected in the urine of patients afflicted with many different types of cancer, including prostate cancer. The effects of p53, NF-κB and the androgen receptor (AR) on the expression of NGAL was examined in four prostate cancer cell lines. Prostate cancer cell lines that are AR negative and expressed either mutant or no p53 (DU145 and PC3) displayed higher levels of NGAL expression compared to the prostate cancer cell lines (LNCaP and 22Rv-1) which are AR positive and express wild type (WT) p53. Introduction of WT-p53 into the PC3 prostate cancer cell line, resulted in reduction of the levels of NGAL expression. Conversely, introduction of dominant negative (DN) p53 or a retroviral construct expressing NF-κB into LNCaP cells increased NGAL expression. NGAL expression had functional effects on the ability of the cells to form colonies in soft agar. Whereas suppression of WT-53 in LNCaP cells increased NGAL expression, the introduction of WT-p53 suppressed NGAL transcription activity in PC3 prostate cells which normally express high level of NGAL. NF-κB and p53 were determined to regulate NGAL expression by positive and negative mechanisms, respectively. Our data indicate that prostate cancer growth, progression and sensitivity to chemotherapeutic drugs are regulated in part by NGAL and may involve complex interactions between NGAL, MMP9, NF-κB and p53.
Bile salts containing vesicles (bilosomes) represent a portentous vesicular carrier that showed prosperous results in delivering active moieties in the gastrointestinal tract (GIT). In this study, bilosomes were exploited to deliver sulphated polysaccharide-protein complexes of Enteromorpha intestinalis (EHEM) and enhance its activity against hepatocellular carcinoma as well as resist harsh GIT conditions. Bilosomes were prepared using the sodium salt of three different bile acids (cholic, deoxycholic, taurodeoxycholic) and two different non-ionic surfactants (Span 40 and 65). The effects of experimental variables were thoroughly studied to obtain an optimum formulation loading EHEM. The selected formulation (EH-Bilo-2) prepared with sodium cholate and Span 65 displayed nano-sized (181.1±16.80 nm) spherical vesicles with reasonable entrapment efficiency (71.60 ± 0.25%) and controlled release properties; and thus was investigated as anti-hepatocarcinogenic candidate for in vivo studies. Treatment of hepatocellular carcinoma (HCC) bearing rats with EH-Bilo-2 experienced significant decrease in serum α-fetoprotein, endoglin, lipocalin-2 and heat shock protein 70 levels versus the untreated counterparts. Furthermore, the photomicrographs of their liver tissue sections showed focal area of degenerated pleomorphic hepatocytes with fine fibrosis originating from the portal area. Thus, the optimized bilosomal formulation is a promising delegate for tackling hepatocellular carcinoma owing to its powerful anti-cancer and anti-angiogenic activity.
Lipocalin-2 (LCN2) is a secreted adipokine that transports small hydrophobic molecules such as fatty acids and steroids. LCN2 limits bacterial growth by sequestering iron-containing siderophores and in mammalian liver protects against inflammation, infection, injury and other stressors. Because LCN2 modulates hepatic fat metabolism and homeostasis, we performed a comparative profiling of proteins and lipids of wild type (WT) and Lcn2-deficient mice fed either standard chow or a methionine- and choline-deficient (MCD) diet. Label-free proteomics and 2D-DIGE protein expression profiling revealed differential expression of BRIT1/MCPH1, FABP5, HMGB1, HBB2, and L-FABP, results confirmed by Western blotting. Gene ontology enrichment analysis identified enrichment for genes associated with mitochondrial membrane permeabilization and metabolic processes involving carboxylic acid. Measurements of mitochondrial membrane potential, mitochondrial chelatable iron pool, intracellular lipid peroxidation, and peroxisome numbers in primary hepatocytes confirmed that LCN2 regulates mitochondrial and peroxisomal integrity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging identified significant changes to sphingomyelins, triglycerides, and glycerophospholipids in livers of mice fed an MCD diet regardless of LCN2 status. However, two arachidonic acid-containing glycerophospholipids were increased in Lcn2-deficient livers. Thus, LCN2 influences peroxisomal and mitochondrial biology in the liver to maintain triglyceride balance, handle oxidative stress, and control apoptosis.
Objective:
Prostate specific antigen (PSA) with digital rectal examination is used for diagnosis of prostate cancer (PCa), where definite diagnosis can only be made by prostate biopsy. Recently neutrophil gelatinase-associated lipocalin (NGAL), a lipocalin family member glycoprotein, come into prominence as a cancer biomarker. This study is aimed to test serum NGAL as a diagnostic biomarker for PCa and discriminate PCa from benign prostatic hyperplasia (BPH).
Material and methods:
In this prospective study, 90 patients who underwent transrectal ultrasound-guided 12-core prostate biopsy between May 2015 and September 2015, were evaluated. Histopathologically diagnosed 45 PCa and 45 BPH patients were enrolled in this study. Serum NGAL and PSA levels of all participants were measured, then these data were evaluated by statistical programs.
Results:
When sensitivity fixed to 80% specificity of NGAL was better than PSA (49%, 31% respectively). Receiver operating characteristic (ROC) curve analysis showed that NGAL alone or its combined use with PSA have better area under curve (AUC) results than PSA alone (0.662, 0.693, and 0.623 respectively).
Conclusion:
In conclusion NGAL gave promising results such as increased sensitivity and a better AUC values in order to distinguish PCa from BPH. NGAL showed a potential to be a non-invasive biomarker which may decrease the number of unnecessary biopsies.
Various, diverse molecules contribute to the tumor microenvironment and influence invasion and metastasis. In this review, the roles of neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) in the tumor microenvironment and sensitivity to therapy will be discussed. The lipocalin family of proteins has many important functions. For example when NGAL forms a complex with MMP-9 it increases its stability which is important in cancer metastasis. Small hydrophobic molecules are bound by NGAL which can alter their entry into and efflux from cells. Iron transport and storage are also influenced by NGAL activity. Regulation of iron levels is important for survival in the tumor microenvironment as well as metastasis. Innate immunity is also regulated by NGAL as it can have bacteriostatic properties. NGAL and MMP-9 expression may also affect the sensitivity of cancer cells to chemotherapy as well as targeted therapy. Thus NGAL and MMP-9 play important roles in key processes involved in metastasis as well as response to therapy. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance.
Copyright © 2015. Published by Elsevier B.V.
The lipocalins were originally classified as a widespread group of transport proteins for small hydrophobic molecules. Although they only share a limited sequence homology their 3D fold is conserved. This group of proteins has been implicated in a multitude of biological processes that most often become visible during disease formation. Lipocalin 2 (LCN2) serves as a siderocalin and protects against bacterial infections. In the liver, LCN2 expression is upregulated during inflammation and in response to cellular stress evolving protective effects during acute and chronic injury. LCN2 was shown to act as an adipokine in the pathogenesis of nonalcoholic fatty liver disease and in control of brown adipose tissue activation. In a nutritional model of nonalcoholic steatohepatitis, LCN2 was identified as a key factor that controls the expression of the perlipin 5 regulating cellular lipid droplet formation. We here summarize experimental and clinical findings linking LCN2 to fatty liver disease.
Background:
Metastasis is the primary cause of prostate cancer (PCa) lethality and poses a huge clinical obstacle. Lipocalin 2 (LCN2), a member of the lipocalin family, is aberrantly expressed in some human cancers and has been implicated in the progression of some tumors. However, the role of LCN2 in the metastatic capacity of prostate cancer (PCa) is poorly understood.
Methods:
LCN2 expression was examined by RT-qPCR and/or immunoblotting in human prostate tissue specimens and prostate cancer cell lines LNCaP, C4-2, 22RV1, PC3, DU-145, and PC3MM2. LCN2 protein level in human serum samples was determined by ELISA. Lentiviruses-mediated over-expression of LCN2 and knockdown of LCN2 was conducted to evaluate the role of LCN2 in cell migratory and invasive capacities of prostate cancer cells. Cell migration and invasion was examined by transwell chamber assay. Knockdown of SLUG by lentivirus was performed to investigate its role in LCN2-promoted cell migration and invasion in vitro (22RV1 cell line) and metastasis in vivo (tail vein metastasis assay in nude mice). Role of ERK signaling in LCN2-mediated up-regulation of SLUG was assayed by using ERK inhibitor U0126.
Results:
We confirmed that LCN2 levels were correlated positively with invasive prostate cancer in human tissue and serum samples, and were also consistently associated with the invasive capacity of prostate cancer cell lines. The over-expression of LCN2 in 22RV1 cells (not highly invasive) promoted the epithelial-mesenchymal transition (EMT), increasing cell motility and invasiveness, while the knockdown of LCN2 in PC3 cells (highly invasive) inhibited EMT, decreasing cell motility and invasiveness. Among the multiple EMT transcription factors, LCN2 specifically induces the expression of SLUG, which was shown here to be required for the LCN2-induced increase in the invasive capacity of prostate cancer cells both in vitro and in vivo. Mechanistically, LCN2 promoted SLUG expression via activating ERK signaling pathway.
Conclusion:
LCN2 plays an important role in promoting cell migration and invasion of prostate cancer by inducing EMT through the ERK/SLUG axis. Therefore, targeted inhibition of LCN2 may represent a therapeutic strategy to prevent the metastasis of prostate cancer.
Prognosis in patients with lung cancer is poor. Neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) are proteins involved in the invasion and metastases of cancer. The objective of this study is to determine if there is a relationship between tumor expression of NGAL and MMP-9 in lung adenocarcinoma patients with prognosis and overall survival. Retrospective analysis was made of patients with lung adenocarcinoma treated at Medica Sur Hospital between 2005 and 2013. Tumor tissue was analyzed for NGAL and MMP-9 expression by immunohistochemistry. We identified 41 patients. Mean overexpression in tumoral tissue of NGAL was 70 % and 30 % for MMP-9. Univariate analysis revealed that prognostic factors associated with overall survival (OS) were NGAL expression and stage at diagnosis. Median OS for NGAL expression <70 % was 45.7 months (95 % CI; 15.2–76.2) and for patients with ≥70 % 4.6 months (95 % CI; 0.5–18.8; P < 0.0001), and for stage at diagnosis (stages I and II mean not reached), stage III mean OS 15.57 months (95 % CI; 9.8–21.2) and stage IV 9.6 months (95 % CI; 0.8–18.4. P = 0.002). No differences in OS were found for expression of MMP-9. Multivariate analysis revealed significance for OS in NGAL expression (HR 5.01 [95 % CI; 1.68–14.93] P = 0.004) and stage at diagnosis (HR 2.05 [95 % CI 1.30–3.22] P = 0.002). Tumoral tissue expression of NGAL ≥70 % confers a worse prognosis compared to those who did not. NGAL is an independent prognostic factor of stage at diagnosis.
We investigated urinary nerve growth factor (NGF) as a novel urinary biomarker for high-grade prostate cancer (PCa).
After institutional review board approval for a prospective pilot study, we enrolled men at the preoperative visit before robotic-assisted radical prostatectomy. Demographics, urinary flow parameters, and urine samples were collected. Urinary NGF and urinary creatinine were obtained in the translational science laboratory. Pathologic and postoperative demographics were collected after surgery. NGF is the primary outcome variable (dependent variable). The pathologic Gleason score (ordinal variable≤6, 7, and ≤8) served as an independent grouping variable. Multivariate analysis using a general linear model was conducted to investigate associations between independent variables and NGF (dependent variable) after adjusting for urinary concentration and volume.
We enrolled and analyzed urine samples and pathologic data from 115 subjects. Patient pathology included 24% (n = 28) Gleason score 6 or less, 68% (n = 78) Gleason score 7, and 8% (n = 9) Gleason score 8 or greater. Perineural invasion was more prevalent in higher-grade disease (P<0.001). The median NGF level was 24.1pg/ml (range: 0.16-270.5pg/ml) and was transformed to the log base 10 scale. Total bladder volume, urinary creatinine level, prostate-specific antigen level, and diabetes were correlated with the Log NGF. In a general linear model, adjusting for bladder volume and urinary creatinine, increasing Log10 NGF was associated with higher Gleason score (Gleason category≤6, 7, and≥8; P = 0.003).
Urinary NGF may be a biomarker for higher-grade PCa. Our pilot study suggests further investigation is warranted to determine whether urinary NGF could provide unique additional information in patients with PCa.
Background:
Lipocalin-2 (LCN2) is a member of the lipocalin superfamily, and it has an important role in the regulation of cellular oncogenesis and apoptosis. However, the role for LCN2 in prostate cancer remains unclear.
Method:
LCN2 expression has been determined by Western blotting, qRT-PCR, and immunohistochemistry in the human prostate cell lines PC3, DU145, LNCaP, and 22Rv, and in human prostate tissue array. In this study, we identified shRNA-LCN2 to determine the role of LCN2 in prostate-cancer cell proliferation, migration, and invasion. Cell proliferative ability was measured by MTT, colony-formation, and cell-cycle analysis. The role of LCN2 in prostate-cancer cell migration and invasion was analyzed by cell-migration assay and Matrigel invasion assay. The effect of LCN2 knockdown on prostate tumor growth was assessed in a subcutaneous xenograft model.
Results:
LCN2 protein and mRNA expression are higher in PC3 and DU145 cells than in LNCaP and 22Rv cells, and prostate cancer tissue correlated significantly with tumor differentiation (P < 0.017) and Gleason's grade (P < 0.02). LCN2 knockdown in PC3 and DU145 cells decreased cell proliferation, colony formation, cell cycle arrest, migration, and invasion. Conversely, LCN2 overexpression in 22Rv cells produced the opposite effect. Subcutaneous xenografts in mice models showed decreased tumor growth in the LCN2-knockdown mice.
Conclusions:
Our results suggest that LCN2 might play an important role in regulation of proliferation and invasion of human prostate cancer, and that it can be a valuable marker of prostate cancer progression.
Matrix metalloproteinases (MMPs) play a central role in tumor invasion and metastasis. Increased expression of MMPs occurs during development of hepatocellular carcinoma (HCC) in humans following infection with hepatitis B virus (HBV). Woodchucks are used as an animal model for hepadnavirus-induced HCC. All woodchucks infected chronically with woodchuck hepatitis virus (WHV), a virus that is closely related to HBV, develop HCC. In the present study MMPs and related molecules were investigated in woodchucks to better understand the mechanisms of extracellular matrix remodeling in HCC. Three groups of samples were studied: liver and HCC tissues from animals infected with WHV and age- and gender-matched normal liver from animals not infected with WHV. New partial gene sequences for woodchuck MMP-2, MMP-7, and MMP-9 as well as their inhibitors NGAL, TIMP-1, and TIMP-2 were identified and used for determination of expression levels in liver and HCC by qRT-PCR. Compared to liver of WHV-naïve woodchucks, high levels of MMP-1, MMP-2, MMP-7, NGAL, and TIMP-1 were detected in liver of animals infected with WHV. However, no differences were found for TIMP-2. MMP-9 expression was higher in HCC than in liver of animals not infected with WHV. Immunohistochemical staining demonstrated that MMP-9 immunoreactivity was most intense in HCC, correlating with the progression of liver disease. Upregulation of MMP-9 in HCC was confirmed by Western blotting and zymography analysis. Furthermore, the activity of woodchuck MMPs was suppressed by BiPS, a common inhibitor of mammalian MMPs. These results suggest the use of MMP inhibitors as a potential HCC treatment strategy that could be explored in woodchucks. J. Med. Virol. 9999:XX-XX, 2013. © 2013 Wiley Periodicals, Inc. J. Med. Virol. © 2013 Wiley Periodicals, Inc.
Iron is an essential nutrient that facilitates cell proliferation and growth. However, iron also has the capacity to engage in redox cycling and free radical formation. Therefore, iron can contribute to both tumour initiation and tumour growth; recent work has also shown that iron has a role in the tumour microenvironment and in metastasis. Pathways of iron acquisition, efflux, storage and regulation are all perturbed in cancer, suggesting that reprogramming of iron metabolism is a central aspect of tumour cell survival. Signalling through hypoxia-inducible factor (HIF) and WNT pathways may contribute to altered iron metabolism in cancer. Targeting iron metabolic pathways may provide new tools for cancer prognosis and therapy.
Aim of the study was to investigate the value of serum and bile neutrophil gelatinase-associated lipocalin (NGAL) for distinguishing malignant strictures caused by cholangiocarcinoma (CCA) or pancreatic cancer from benign biliary strictures. The study was performed prospectively on patients admitted for endoscopic or radiologic biliary decompression. Forty patients with dilated biliary ducts, including 16 cases of CCA, 6 cases of pancreatic cancer, and 18 cases of benign biliary stricture were enrolled. Their sera and bile were collected to measure NGAL. Routine biochemistry including measurement of serum levels of carbohydrate antigens (CA) 19-9 and carcinoembryonic antigen (CEA) was also performed. The serum CA19-9, serum CEA, and bile NGAL levels were significantly increased in patients with malignant strictures as compared with patients with benign biliary diseases. Serum NGAL had no significant value for discriminating between malignant and benign biliary strictures. Bile NGAL levels had a receiver characteristic area under the curve of 0.74, sensitivity 77.3, and specificity 72.2% for discriminating between pancreatobiliary cancer and benign biliary diseases. Bile NGAL and serum CA19-9 were independent parameters and their combined use improved diagnostic accuracy (sensitivity 91%, negative predictive value 85.7%). We conclude that measurement of biliary, but not serum NGAL, may differentiate malignant pancreatobiliary from benign biliary strictures, serving as a complementary biomarker for serum CA19-9.
Lung cancer is the leading worldwide cause of cancer deaths. Smoking is the dominant cause of lung cancer and smoking cessation is the established method to reduce lung cancer mortality. While lung cancer risk is reduced in former smokers, they have a lifelong increase in risk, compared to never-smokers. Novel chemoprevention strategies, such as oral or inhaled prostacyclin analogs, hold promise for these subjects. Low-dose spiral computed tomography screening reduced lung cancer mortality by 20% in high-risk heavy smokers older than 50 years. However, the high false-positive rate (96%) means that screened patients required controlled follow-up in experienced centers. An increasing percentage of patients with advanced lung cancer have molecular drivers in genes for which oral tyrosine kinase inhibitors have been developed.
Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, has been found to be overexpressed in a variety of tumors, including lung adenocarcinomas. However, the mechanism by which NGAL expression is regulated in lung carcinoma needs further evaluation. In this study, immunohistochemistry was employed to analyze the expression of NGAL in lung carcinoma tissue samples, including lung squamous carcinomas, adenocarcinomas, adenosquamous carcinomas and bronchial alveolar cell carcinomas. The results showed that NGAL was expressed in 82.61% (19/23) of the samples. RT-PCR and immunofluorescent staining showed that NGAL was localized to the cytoplasm in lung carcinoma cell lines. To explore the transcriptional regulation mechanism of NGAL basal expression in lung carcinoma, a 1515-bp fragment (-1431 to +84) of the NGAL promoter region was cloned and a series of deletion and mutation constructs were generated. These constructs were analyzed using the luciferase reporter assay. The results indicated that the cis-acting elements important for the basal activity of NGAL transcription were likely located between -152 and -141. Further analysis using site-directed mutagenesis and the luciferase reporter assay suggested that the C/EBP binding sites were responsible for the activity of the NGAL promoter. Finally, the binding ability and specificity of the transcription factors were determined by electrophoretic mobility-shift assay (EMSA). The results showed that C/EBPβ was able to bind to the -152 and -141 segments. Taken together, these findings suggest that NGAL is expressed in lung carcinomas and that NGAL expression is mediated by the binding of C/EBPβ to the -152 and -141 segment of the NGAL promoter.
The Northeastern region of Thailand is well known to have high incidence of bile duct cancer known as cholangiocarcinoma. So there is a continued need to improve diagnosis and treatment, and discovery of biomarkers for early detection of bile duct cancer should greatly improve treatment outcome for these patients. The secretome, a collection of proteins secreted from cells, is a useful source for identifying circulating biomarkers in blood secreted from cancer cells. Here a Hollow Fiber Bioreactor culture system was used for enrichment of cholangiocarcinoma secretomes, since this culture system mimics the dense three-dimensional microenvironment of the tumor found in vivo. Two-dimensional fluorescence difference gel electrophoresis using a sensitive Fluor saturation dye staining, followed by LC/MS/MS, was used to compare protein expression in the secretomes of cells cultured in the Hollow Fiber system and cells cultured in the monolayer culture system. For the first time, the 2D-patterns of cholangiocarcinoma secretomes from the two culture systems could be compared. The Hollow Fiber system improved the quality and quantity of cholangiocarcinoma secreted proteins compared to conventional monolayer system, showing less interference by cytoplasmic proteins and yielding more secreted proteins. Overall, 75 spots were analyzed by LC/MS/MS and 106 secreted proteins were identified. Two novel secreted proteins (C19orf10 and cystatin B) were found only in the Hollow Fiber system and were absent from the traditional monolayer culture system. Among the highly expressed proteins, 22 secreted soluble proteins were enriched by 5 fold in Hollow Fiber system compared to monolayer culture system. The Hollow Fiber system is therefore useful for preparing a wide range of proteins from low-abundance cell secretomes.
Aim:
Neutrophil gelatinase-associated lipocalin (NGAL) and its cell surface receptor, NGALR, have been implicated in tumorigenesis and tumor progression of various human malignant neoplasms. In particularly, it has been demonstrated that NGAL is overexpressed in hepatocellular carcinoma (HCC) tissues and closely associated with the proliferation and invasion of HCC cells. The aim of this study was to investigate the clinical significance of NGAL and NGALR in HCC.
Methods:
Expression of NGAL and NGALR was evaluated by immunohistochemistry in tumor tissues from 138 patients who underwent curative resection of HCC. The association of NGAL or NGALR expression with the clinicopathologic features was analyzed. Univariate and multivariate analyses were performed to evaluate the prognostic value of NGAL and/or NGALR expression for HCC patients.
Results:
The expression levels of NGAL and NGALR were both up-regulated in HCC tissues, and to be associated with vascular invasion (both P=0.03), TNM stage (both P=0.004), and tumor recurrence (both P<0.001). A positive correlation between expression of the two markers was also observed (r=0.89; P<0.001). Additionally, survival analysis showed that high expression of NGAL or NGALR was significantly associated with poor prognosis for patients with HCC (both P=0.003). Patients with high expression of both NGAL and NGALR had a shorter overall survival (P<0.001) than those with low expression of both. Furthermore, multivariate analysis showed both NGAL and NGALR were independent predictors of overall survival.
Conclusion:
Our data demonstrate for the first time that the up-regulations of NGAL and NGALR expression in HCC were both significantly correlated with unfavorable clinicopathologic features and independent poor prognostic factor for overall survival in patients. These findings suggest that NGAL and NGALR expression might be served as novel prognostic factors and potential therapeutic targets in HCC.
Lipocalin 2 (LCN2) is a secreted, iron-binding glycoprotein that is abnormally expressed in some malignant human cancers. However, the roles of LCN2 in hepatocellular carcinoma (HCC) cells are unknown. In this study, we suggested the LCN2 and LCN2R were weak detected in the HCC cell lines, LCN2 and LCN2R were found to be down-regulated in tumor tissues in 16 HCC patients. MTT, DAPI, TUNEL, and flow cytometry analyses revealed that LCN2 overexpression dramatically inhibited cell viability, induced apoptosis features of cell-cycle arrest in sub-G1 phase, in DNA fragmentation, and in condensation of chromatin in Huh-7 and SK-Hep-1 cells. Western blots were used to detect the activation of caspase, pro-apoptosis, and anti-apoptosis protein expression in overexpress-LCN2 HCC cells. LCN2-induced apoptosis was characterized by cleavage of caspase-9, -8, -3, and PARP protein, and a reduction in the mitochondrial membrane potential (MMP). Furthermore, LCN2 also enhanced the down-regulated Bcl-2 and up-regulated the expression of Bax. In addition, our experiments with caspase inhibitors LEHD-FMK and IETD-FMK prevent LCN2-induced apoptosis. We also demonstrated that treatment of overexpress-LCN2 HCC cells with the LCN2 neutralized antibody also significantly attenuated LCN2-induced cell apoptosis. These findings indicate that LCN2 overexpression can effectively induce apoptosis of HCC cells and may be used as a potent therapy against human HCC.
Distant metastasis is one of the leading causes of lung cancer death. Detecting the early-stage molecular alternations in primary tumors, such as gene expression differences, provides a "prognostic" value to the precaution of tumor metastasis. The aim of this article is to screen and identify the metastasis-related genes in human squamous cell lung carcinoma. Primary tumor tissues of nine patients with subsequent metastasis and eight patients without metastasis were selected to perform the gene microarray experiment. GO and pathway analyses were used to determine the differentially expressed genes. Two identified genes were further validated by real-time quantitative reverse transcription polymerase chain reaction (PCR) (real-time qRT-PCR). Two hundred and thirty-eight differentially expressed genes were detected in gene chip experiment, including 51 up-regulated genes and 187 down-regulated genes. These genes were involved in several cellular processes, including cell adhesion, cell cycle regulation, and apoptosis. GO analysis showed that the differentially expressed genes participated in a wide ranging of metastasis-related processes, including extracellular region and regulation of liquid surface tension. In addition, pathway analysis demonstrated that the differentially expressed genes were enriched in pathways related to cell cycle and Wnt signaling. Real-time qRT-PCR validation experiment of LCN2 and PDZK1IP1 showed a consistent up-regulation in the metastasis group. The metastasis of human squamous cell lung carcinoma is a complex process that is regulated by multiple gene alternations on the expression levels. The 238 differentially expressed genes identified in this study presumably contain a core set of genes involved in tumor metastasis. The real-time qRT-PCR results of PDZK1IP1 and LCN2 validated the reliability of this gene microarray experiment.
Cancer is one of the leading causes of death, and there is an urgent need for new biomarkers and therapeutic targets. The progesterone receptor membrane component 1 (Pgrmc1) protein is upregulated in multiple types of cancer, and Pgrmc1 is required for tumor cell proliferation, motility and tumor formation in vivo. Furthermore, a small molecule inhibitor of Pgrmc1 suppressed the growth of lung, breast and cervical cancer cell lines. Recently, Pgrmc1 was identified as the sigma-2 receptor, a putative type of opioid receptor, and sigma-2 receptors are induced in cancers. However, Pgrmc1 shares no homology with known opioid or hormone receptors but is related to cytochrome b(5), and Pgrmc1 binds to heme and has reducing activity. In this study, we have analyzed Pgrmc1 levels in clinical tumor samples from squamous cell lung cancers (SCLC) and lung adenocarcinomas compared to corresponding nonmalignant tissue. Pgrmc1 levels increased significantly (p ≤ 0.05) in 12/15 SCLC samples and was elevated in poorly differentiated tumors. Pgrmc1 was highly expressed in SCLC cell lines, and SCLC cell survival was inhibited by siRNA knockdown of Pgrmc1 or the Pgrmc1 inhibitor AG-205. In adenocarcinomas, 6/15 tumors significantly had elevated Pgrmc1 levels, which correlated with patient survival. Pgrmc1 localizes to secretory vesicles in cancer cells, and Pgrmc1 was secreted by lung cancer cells. Furthermore, Pgrmc1 was significantly elevated in the plasma of lung cancer patients compared to noncancer patients. Together, the results demonstrate that Pgrmc1 is a potential tumor and serum biomarker, as well as a therapeutic target, for lung cancer.
Prostate-specific antigen (PSA) is a protein produced by the prostate, and this protein may be elevated for several reasons, including prostatitis, benign prostatic hypertrophy and/or cancer. PSA is not cancer-specific, cannot be used as a cancer marker and it has been demonstrated that there is no level of PSA that is definitive for prostate cancer. The value of the PSA test varies when used for screening, diagnosis, prognosis or as a signal of disease recurrence. Misuse of the test for screening has created unnecessary anxiety and costs, and has led to the significant overdiagnosis and overtreatment of men. More important than whether or not to screen is how one acts upon the data from a single test; with the exception of extremely high double- or triple-digit levels of PSA, it is prudent only to use a single PSA determination as a baseline, with biopsy and cancer treatment reserved for those with significant PSA changes over time, or for those with clinical manifestations mandating immediate therapy. Using the PSA test to monitor disease progression or recurrence is appropriate, provided one understands that absolute levels of PSA are rarely meaningful; it is the relative change in PSA levels over time that provides insight, but not definitive proof of a cancerous condition necessitating therapy. PSA secretion is under hormonal control and thus PSA levels may be affected differently by the type of drug therapy, by the stage of a patients' disease, and by genetic factors suggesting some men are 'high PSA producers'. Until a validated alternative test for prostate cancer is found and adopted, the current flawed PSA test needs to be used more judiciously and not used for routine screening as studies have demonstrated that screening, as defined, does not lead to a reduction in patient mortality. All men, their families and their physicians need to understand the significant limitations of PSA testing.