CTSD

Gene Summary

Gene:CTSD; cathepsin D
Aliases: CPSD, CLN10, HEL-S-130P
Location:11p15.5
Summary:This gene encodes a lysosomal aspartyl protease composed of a dimer of disulfide-linked heavy and light chains, both produced from a single protein precursor. This proteinase, which is a member of the peptidase C1 family, has a specificity similar to but narrower than that of pepsin A. Transcription of this gene is initiated from several sites, including one which is a start site for an estrogen-regulated transcript. Mutations in this gene are involved in the pathogenesis of several diseases, including breast cancer and possibly Alzheimer disease. [provided by RefSeq, Jul 2008]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:cathepsin D
HPRD
Source:NCBIAccessed: 27 February, 2015

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 27 February 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CTSD (cancer-related)

Sun Z, Dong J, Zhang S, et al.
Identification of chemoresistance-related cell-surface glycoproteins in leukemia cells and functional validation of candidate glycoproteins.
J Proteome Res. 2014; 13(3):1593-601 [PubMed] Related Publications
Chemoresistance remains the most significant obstacle to successful chemotherapy for leukemia, and its exact mechanism is still unknown. In this work, we used the cell-surface capturing method together with quantitative proteomics to investigate differences in the glycoproteomes of adriamycin-sensitive and adriamycin-resistant leukemia cells. Two quantitative methods, isotopic dimethyl labeling and SWATH, were used to quantify glycoproteins, and 35 glycoproteins were quantified by both methods. High correlation was observed between the glycoproteins quantified by the above two methods, and 15 glycoproteins displayed a consistent significant change trend in both sets of quantitative results. These 15 proteins included classical multidrug resistance-related glycoproteins such as ABCB1 as well as a set of novel glycoproteins that have not previously been reported to be associated with chemoresistance in leukemia cells. Further validation with quantitative real-time PCR and Western blotting confirmed the proteomic screening results. Subsequent functional experiments based on RNA interference technology showed that CTSD, FKBP10, and SLC2A1 are novel genes that participate in the acquisition and maintenance of the adriamycin-resistant phenotype in leukemia cells.

Nait Achour T, Sentis S, Teyssier C, et al.
Transcriptional repression of estrogen receptor α signaling by SENP2 in breast cancer cells.
Mol Endocrinol. 2014; 28(2):183-96 [PubMed] Free Access to Full Article Related Publications
Estrogen receptors (ERs) are ligand-activated transcription factors involved in many physiological and pathological processes, including breast cancer. Their activity is fine-tuned by posttranslational modifications, notably sumoylation. In the present study, we investigated the role of the small ubiquitin-related modifier (SUMO) protease, SUMO1/sentrin/suppressor of Mif 2-specific peptidase 2 (SENP2), in the regulation of ERα activity. We first found SENP2 to significantly repress estradiol-induced transcriptional activity in breast cancer cells (MCF7 and T47D). This effect was observed with a reporter plasmid and on endogenous genes such as TFF1 and CTSD, which were shown to recruit SENP2 in chromatin immunoprecipitation experiments. Using glutathione S-transferase pull-down, coimmunoprecipitation and proximity ligation assays, SENP2 was found to interact with ERα and this interaction to be mediated by the amino-terminal region of the protease and the hinge region of the receptor. Interestingly, we demonstrated that ERα repression by SENP2 is independent of its SUMO protease activity and requires a transcriptional repressive domain located in the amino-terminal end of the protease. Using small interfering RNA assays, we evidenced that this domain recruits the histone deacetylase 3 (HDAC3), to be fully active. Furthermore, using both overexpression and knockdown strategies, we showed that SENP2 robustly represses estrogen-dependent and independent proliferation of MCF7 cells. We provided evidence that this effect requires both the proteolytic and transcriptional activities of SENP2. Altogether, our study unravels a new property for a SUMO protease and identifies SENP2 as a classical transcription coregulator.

Cui Y, Xie S, Luan J, et al.
Quantitative proteomics and protein network analysis of A549 lung cancer cells affected by miR-206.
Biosci Trends. 2013; 7(6):259-63 [PubMed] Related Publications
MiR-206 acts as a potential tumor suppressor during carcinogenesis and a regulatory factor in osteoblasts differentiation, but its modulatory mechanism remains unclear. In this study, we used a quantitative proteomics method, difference gel electrophoresis (DIGE), to profile the protein variation in A549 lung cancer cells with and without miR- 206 transfection. We identified a total of 17 differently expressed proteins including 5 up-regulated and 12 down-regulated proteins affected by miR-206 in A549 cells. We further constructed a protein network linked 17 differently expressed proteins with 106 computationally predicted miR-206 targets, and identified 8 "hub" genes (CALR, CTSD, ENO1, HSPA5, CDC42, HSPD1, POLA1, and SMARCA4) within the network, which may represent important miR-206 functional gene targets. In conclusion, in this study, we identified several candidate functional target genes for miR-206, which is helpful to further explore its mechanisms during carcinogenesis and osteogenesis, and we also proposed a novel proteomic strategy to identify functionally important gene targets for microRNA.

Mikulová V, Cabiňaková M, Janatková I, et al.
Detection of circulating tumor cells during follow-up of patients with early breast cancer: Clinical utility for monitoring of therapy efficacy.
Scand J Clin Lab Invest. 2014; 74(2):132-42 [PubMed] Related Publications
INTRODUCTION: Circulating tumor cells (CTCs) detection prior to and during therapy is considered as an independent and strong prognostic marker. The present study was designed to isolate and characterize CTCs in peripheral blood of an early breast cancer (BC) patient as a biomarker for monitoring treatments efficacy.
MATERIALS AND METHODS: In total, 54 early breast cancer patients undergoing neoadjuvant and/or adjuvant chemotherapy regimens were enrolled into a prospective study. CTC detection in blood was performed by AdnaTest BreastCancer(™) (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. Additionally, cDNA from isolated CTCs and PBMC was used for qPCR gene expression analysis of TOP1, TOP2A, CTSD, ST6, CK19 and reference gene actin.
RESULTS: We found that CTCs can be detected in the peripheral blood of approximately 31% of early stage breast cancer patients. The presence of CTCs was detected in 36% ER positive, 32% PR positive and 30% HER2 positive patients. We found no correlation between CTCs and tumor size, tumor grade, histological grade and receptor status. Only 7% of all patients remained CTCs positive after adjuvant therapy. Gene expression analysis revealed a particular heterogeneity of the studied genes.
CONCLUSIONS: In conclusion, CTC detection may be a promising early marker of disease progression potentially enhancing the difficult therapeutic decisions. Further studies should, however, clearly demonstrate its utility for both the prediction of outcome and monitoring the effect of treatment.

Crabtree D, Dodson M, Ouyang X, et al.
Over-expression of an inactive mutant cathepsin D increases endogenous alpha-synuclein and cathepsin B activity in SH-SY5Y cells.
J Neurochem. 2014; 128(6):950-61 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α-synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α-synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α-synuclein degradation, as evidenced by the accumulation of endogenous α-synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy-lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over-expressing wild-type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α-synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α-synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

Qiao S, Tao S, Rojo de la Vega M, et al.
The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death.
Autophagy. 2013; 9(12):2087-102 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A, E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells.

Shu G, Mi X, Cai J, et al.
Brucine, an alkaloid from seeds of Strychnos nux-vomica Linn., represses hepatocellular carcinoma cell migration and metastasis: the role of hypoxia inducible factor 1 pathway.
Toxicol Lett. 2013; 222(2):91-101 [PubMed] Related Publications
Brucine is an alkaloid derived from the seeds of Strychnos nux-vomica Linn. which have long been used as a traditional medicine for the treatment of hepatocellular carcinoma (HCC) in China. HCC prognosis can be greatly influenced by metastasis. There has thus far been little research into brucine as a source of anti-metastasis activity against HCC. In this study, we revealed that brucine dramatically repressed HepG2 and SMMC-7721 HCC cell migration with few cytotoxic effects. Hypoxia inducible factor 1 (HIF-1) is a key transcription factor mediating cell migration and invasion. Brucine suppressed HIF-1-dependent luciferase activity in HepG2 cells. The transcriptions of four known HIF-1 target genes involved in HCC metastasis, i.e., fibronectin, matrix metallopeptidase 2, lysyl oxidase, and cathepsin D, were also attenuated after brucine treatment. Experiments in vivo showed that an intraperitoneal injection of 5 and 15 mg/kg of brucine resulted in dose-dependent decreases in the lung metastasis of H22 ascitic hepatoma cells. Moreover, a dosage of brucine at 15 mg/kg exhibited very low toxic effects to tumor-bearing mice. Consistently, brucine downregulated expression levels of HIF-1 responsive genes in vivo. Our current study demonstrated the capacity of brucine in suppressing HCC cell migration in vitro and lung metastasis in vivo. The inhibition of the HIF-1 pathway is implicated in the anti-metastasis activity of brucine.

Pernemalm M, De Petris L, Branca RM, et al.
Quantitative proteomics profiling of primary lung adenocarcinoma tumors reveals functional perturbations in tumor metabolism.
J Proteome Res. 2013; 12(9):3934-43 [PubMed] Related Publications
In this study, we have analyzed human primary lung adenocarcinoma tumors using global mass spectrometry to elucidate the biological mechanisms behind relapse post surgery. In total, we identified over 3000 proteins with high confidence. Supervised multivariate analysis was used to select 132 proteins separating the prognostic groups. Based on in-depth bioinformatics analysis, we hypothesized that the tumors with poor prognosis had a higher glycolytic activity and HIF activation. By measuring the bioenergetic cellular index of the tumors, we could detect a higher dependency of glycolysis among the tumors with poor prognosis. Further, we could also detect an up-regulation of HIF1α mRNA expression in tumors with early relapse. Finally, we selected three proteins that were upregulated in the poor prognosis group (cathepsin D, ENO1, and VDAC1) to confirm that the proteins indeed originated from the tumor and not from a stromal or inflammatory component. Overall, these findings show how in-depth analysis of clinical material can lead to an increased understanding of the molecular mechanisms behind tumor progression.

Wu X, Hu A, Zhang M, Chen Z
Effects of Rab27a on proliferation, invasion, and anti-apoptosis in human glioma cell.
Tumour Biol. 2013; 34(4):2195-203 [PubMed] Related Publications
This study aims to investigate the relationship between Rab27a and the characteristics of glioma cell U251 such as proliferation, apoptosis, and invasion and to provide an experimental basis for future therapy in human glioma. Recombinant plasmid of pcDNA3.1-Rab27a was constructed and transfected into U251 cells with the help of Lipofectamine™2000. The expression of Rab27a was detected by Western blot. Cell viability, cell cycle, cell apoptosis, and cell migration were analyzed, respectively, by (3-(4,5)-dimethylthi-azol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay, flow cytometry, and Transwell invasion chamber methods. Meanwhile, the effect of Rab27a on secretion of cathepsin D in U251 cells was also examined. With the help of luciferase reporter assay system, the relationship between miR-124 and gene Rab27a expression was explored. Western blot showed that the expression of Rab27a was significantly increased in pcDNA3.1-Rab27a transfection group (p < 0.01) and that was significantly decreased in Rab27a-shRNA transfection group (p < 0.01) compared with control group. MTT assay, flow cytometry, and Transwell invasion chamber experiment indicated that cell viability (p < 0.01), proliferation index (p < 0.05), and invasion ability (p < 0.01) were improved significantly in pcDNA3.1-Rab27a transfection group compared with control group and that cell viability (p < 0.01), proliferation index (p < 0.05), and invasion ability (p < 0.01) were reduced markedly in Rab27a-shRNA transfection group compared with control group. The apoptosis analysis by flow cytometry demonstrated that the ratio of apoptosis in pcDNA3.1-Rab27a transfection group was significantly lower than that in control group (p < 0.05) and the ratio was notably higher in Rab27a-shRNAtransfection group than that in the control group. Cathepsin D activity assay indicated that the release of cathepsin D was enhanced in pcDNA3.1-Rab27a transfection group compared to that in the control group (p < 0.05). Rab27a could increase the glioma cell ability, promote proliferation and invasion, and suppress cell apoptosis. The above-stated effects of Rab27a possibly were exerted by increasing the secretion of cathepsin D and regulated by miR-124. In addition, the inhibition of expression of Rab27a perhaps benefited the therapy for glioma patients.

Liu Z, Chen C, Yang H, et al.
Proteomic features of potential tumor suppressor NESG1 in nasopharyngeal carcinoma.
Proteomics. 2012; 12(22):3416-25 [PubMed] Related Publications
We previously defined the recently revised NESG1 gene as a potential tumor suppressor in nasopharyngeal carcinoma (NPC). Here, we further used proteomics technology to globally examine NESG1-controlled proteins in NPC cells. Twenty-six proteins were found to be deregulated by NESG1 using proteomics analysis while enolase 1 (alpha) (ENO1), heat shock protein 90 kDa beta (Grp94), member 1 (HSP90B1), and cathepsin D (CTSD) proteins were differentially expressed by Western blot. Interestingly, a-enolase (ENO1), an overexpressed gene in NPC, was confirmed as a NESG1-regulated protein in NPC cells. Overexpressed ENO1 not only restored cell proliferation and cell-cycle progression, but also antagonized the regulation of NESG1 to cell-cycle regulators p21 and CCNA1 expression as well as induced the expression of C-Myc, pRB, and E2F1 in NESG1-ovexpressed NPC cells. Real-time PCR and immunohistochemistry analysis showed that NESG1 expression is negatively correlated with ENO1 expression in NPC tissues. Our observations suggest that ENO1 downregulation plays an important role in NESG1-induced growth inhibition of NPC cancer cells.

Pruitt FL, He Y, Franco OE, et al.
Cathepsin D acts as an essential mediator to promote malignancy of benign prostatic epithelium.
Prostate. 2013; 73(5):476-88 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
BACKGROUND: Stromal-epithelial interactions are important in both development and prostate cancer. Stromal changes have been shown to be powerful prognostic indicators of prostate cancer progression and of patient death helping to define lethal versus indolent phenotypes. The specific molecular underpinnings of these interactions are incompletely understood. We investigated whether stromal cathepsin D (CathD) overexpression affects prostate tumorigenesis through a paracrine mechanism.
METHODS: Normal prostate fibroblasts (NPF) were retrovirally transduced to overexpress cyclin D1 (CD1) and were designated NPF(CD1) . Cathepsin D expression was knocked down using shRNA in cancer associated fibroblasts (CAF) and NPF(CD1) . We analyzed these stromal cell lines using immunohistochemistry, Western blot, and tissue recombination.
RESULTS: An examination of human prostate tissue revealed significantly increased stromal staining of CathD in malignant prostate tissue. Overexpression of CD1 in normal prostate fibroblasts (NPF(CD1) ) produced a phenotype similar to, but more moderate than, CAF in a tissue recombination model. Knockdown studies revealed that CathD is required for NPF(CD1) motility and invasive growth in vitro. BPH-1 cell proliferation was found to be induced when cultured with NPF(CD1) conditioned medium, this effect was inhibited when CathD was knocked down in NPF(CD1) cells. Overexpression of CathD in prostate stromal cells induced malignancy in adjacent epithelium, and this transformation was inhibited when stromal CathD expression was knocked down in CAF.
CONCLUSIONS: The study presented here demonstrates increased CathD expression is seen in human CAF. The upregulation of CD1 results in concomitant increases in CathD expression. Elevated CathD expression in the stroma contributes to tumor promotion.

Mimae T, Tsuta K, Maeshima AM, et al.
Cathepsin D as a potential prognostic marker for lung adenocarcinoma.
Pathol Res Pract. 2012; 208(9):534-40 [PubMed] Related Publications
We previously identified cathepsin D as a possible marker for lung adenocarcinoma (AD). The purpose of the present study is to evaluate the correlation between cathepsin D expression and clinicopathological findings or prognosis. We conducted immunohistochemistry (IHC) to assess 150 AD tissues. For these 150 tumors, TTF-1 expression, EGFR and KRAS gene mutations, and ALK rearrangements had already been examined. Cathepsin D expression was detected in 44% (66 of 150, IHC score ≥1+) and 27.3% (41 of 150, IHC score ≥2+). Cathepsin D-positive (IHC score ≥2+) tumors were more poorly differentiated than cathepsin D-negative ones, while all lepidic predominant invasive adenocarcinomas showed no cathepsin D expression. Univariate analysis revealed a poor prognosis for cathepsin D-positive lung AD patients with an IHC score ≥2+ (P=0.044). Cathepsin D expression was more frequent in TTF-1-negative than in TTF-1-positive ADs (P=0.034), and more frequent in ADs with EGFR wild genotype than mutant EGFR (P<0.001). Regarding AD patients with ALK rearrangements, 4 were positive for Cathepsin D, while 2 were negative. Cathepsin D expression is indicated to be a possible prognostic marker for lung AD and to correlate with a more poorly differentiated form.

Sudhir PR, Chen CH, Pavana Kumari M, et al.
Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells.
Mol Cell Proteomics. 2012; 11(10):901-15 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRAS(V12)). Using an OFFGEL peptide fractionation and hydrazide method combined with subsequent LTQ-Orbitrap analysis, we identified 5713 proteins and 608 N-glycosites on 317 proteins in human bronchial epithelial cells. Label-free quantitation of 3058 proteins (≥2 peptides; coefficient of variation (CV) ≤ 20%) and 297 N-glycoproteins (CV ≤ 20%) revealed the differential regulation of 23 proteins and 14 N-glycoproteins caused by activated KRAS, including 84% novel ones. An informatics-assisted IPA-Biomarker® filter analysis prioritized some of the differentially regulated proteins (ALDH3A1, CA2, CTSD, DST, EPHA2, and VIM) and N-glycoproteins (ALCAM, ITGA3, and TIMP-1) as cancer biomarkers. Further, integrated in silico analysis of microarray repository data of lung adenocarcinoma clinical samples and cell lines containing KRAS mutations showed positive mRNA fold changes (p < 0.05) for 61% of the KRAS-regulated proteins, including biomarker proteins, CA2 and CTSD. The most significant discovery of the integrated validation is the down-regulation of FABP5 and PDCD4. A few validated proteins, including tumor suppressor PDCD4, were further confirmed as KRAS targets by shRNA-based knockdown experiments. Finally, the studies on KRAS-regulated N-glycoproteins revealed structural alterations in the core N-glycans of SEMA4B in KRAS-activated human bronchial epithelial cells and functional role of N-glycosylation of TIMP-1 in the regulation of lung adenocarcinoma A549 cell invasion. Together, our study represents the largest proteome and N-glycoproteome data sets for HBECs, which we used to identify several novel potential targets of activated KRAS that may provide insights into KRAS-induced adenocarcinoma and have implications for both lung cancer therapy and diagnosis.

Tang S, Huang W, Zhong M, et al.
Identification Keratin 1 as a cDDP-resistant protein in nasopharyngeal carcinoma cell lines.
J Proteomics. 2012; 75(8):2352-60 [PubMed] Related Publications
Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy of tumors. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cis-diamminedichloroplatinum (CNE2/cDDP) were established from human nasopharyngeal carcinoma (NPC) cell lines CNE2. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and ESI-Q-TOF-MS were performed on protein extracted from CNE2 and CNE2/cDDP cell lines to screen drug resistance-related proteins. Keratin 1 (KRT1), cathepsin D (CTSD) and annexin a5 (ANXA5) were identified as three proteins showing higher expression in CNE2/cDDP compared to CNE2. Furthermore, suppression of KRT1 expression by siRNA resulted in decreased MDR in siRNA-CNE2/cDDP cells. And upregulation of KRT1 could result in increased of drug resistance in NPC cell lines. Taken together, KRT1 protein and its activity levels were higher in cDDP-resistant NPC cell lines compared to their parental cell lines. These data clearly linked KRT1 and cDDP resistance mechanisms. KRT1 could serve as a biomarker for chemotherapy sensitivity of NPC.

Carew JS, Espitia CM, Esquivel JA, et al.
Lucanthone is a novel inhibitor of autophagy that induces cathepsin D-mediated apoptosis.
J Biol Chem. 2011; 286(8):6602-13 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
Cellular stress induced by nutrient deprivation, hypoxia, and exposure to many chemotherapeutic agents activates an evolutionarily conserved cell survival pathway termed autophagy. This pathway enables cancer cells to undergo self-digestion to generate ATP and other essential biosynthetic molecules to temporarily avoid cell death. Therefore, disruption of autophagy may sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis. Chloroquine and its analog hydroxychloroquine are the only clinically relevant autophagy inhibitors. Because both of these agents induce ocular toxicity, novel inhibitors of autophagy with a better therapeutic index are needed. Here we demonstrate that the small molecule lucanthone inhibits autophagy, induces lysosomal membrane permeabilization, and possesses significantly more potent activity in breast cancer models compared with chloroquine. Exposure to lucanthone resulted in processing and recruitment of microtubule-associated protein 1 light chain 3 (LC3) to autophagosomes, but impaired autophagic degradation as revealed by transmission electron microscopy and the accumulation of p62/SQSTM1. Microarray analysis, qRT-PCR, and immunoblotting determined that lucanthone stimulated a large induction in cathepsin D, which correlated with cell death. Accordingly, knockdown of cathepsin D reduced lucanthone-mediated apoptosis. Subsequent studies using p53(+/+) and p53(-/-) HCT116 cells established that lucanthone induced cathepsin D expression and reduced cancer cell viability independently of p53 status. In addition, lucanthone enhanced the anticancer activity of the histone deacetylase inhibitor vorinostat. Collectively, our results demonstrate that lucanthone is a novel autophagic inhibitor that induces apoptosis via cathepsin D accumulation and enhances vorinostat-mediated cell death in breast cancer models.

Johnsen SA, Güngör C, Prenzel T, et al.
Regulation of estrogen-dependent transcription by the LIM cofactors CLIM and RLIM in breast cancer.
Cancer Res. 2009; 69(1):128-36 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
Mammary oncogenesis is profoundly influenced by signaling pathways controlled by estrogen receptor alpha (ERalpha). Although it is known that ERalpha exerts its oncogenic effect by stimulating the proliferation of many human breast cancers through the activation of target genes, our knowledge of the underlying transcriptional mechanisms remains limited. Our published work has shown that the in vivo activity of LIM homeodomain transcription factors (LIM-HD) is critically regulated by cofactors of LIM-HD proteins (CLIM) and the ubiquitin ligase RING finger LIM domain-interacting protein (RLIM). Here, we identify CLIM and RLIM as novel ERalpha cofactors that colocalize and interact with ERalpha in primary human breast tumors. We show that both cofactors associate with estrogen-responsive promoters and regulate the expression of endogenous ERalpha target genes in breast cancer cells. Surprisingly, our results indicate opposing functions of LIM cofactors for ERalpha and LIM-HDs: whereas CLIM enhances transcriptional activity of LIM-HDs, it inhibits transcriptional activation mediated by ERalpha on most target genes in vivo. In turn, the ubiquitin ligase RLIM inhibits transcriptional activity of LIM-HDs but enhances transcriptional activation of endogenous ERalpha target genes. Results from a human breast cancer tissue microarray of 1,335 patients revealed a highly significant correlation of elevated CLIM levels to ER/progesterone receptor positivity and poor differentiation of tumors. Combined, these results indicate that LIM cofactors CLIM and RLIM regulate the biological activity of ERalpha during the development of human breast cancer.

Sagulenko V, Muth D, Sagulenko E, et al.
Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death.
Carcinogenesis. 2008; 29(10):1869-77 [PubMed] Related Publications
High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.

Le Dily F, Métivier R, Guéguen MM, et al.
COUP-TFI modulates estrogen signaling and influences proliferation, survival and migration of breast cancer cells.
Breast Cancer Res Treat. 2008; 110(1):69-83 [PubMed] Related Publications
We previously showed that COUP-TFI interacts with the Estrogen Receptor alpha (ER alpha) to recruit Extracellular signal Regulated Kinases (ERKs) in an Estradiol (E2)-independent manner, resulting in an enhancement of ER alpha transcriptional activity. However, the involvement of COUP-TFI in physiologically relevant functions of ER alpha, such as the mitogenic activity that E2 has on breast cancer cells, remains poorly understood. Here, we first showed that the amounts of COUP-TFI protein are higher in dedifferentiated mammary cell lines (MDA-MB-231) and tumor breast cells as compared to the differentiated MCF-7 cell line and normal breast cells. To evaluate the functional relevance of the COUP-TFI/ER alpha interplay in mammary cells, we generated MCF-7 cells that stably over-express COUP-TFI. We found that the over-expression of COUP-TFI enhances motility and invasiveness of MCF-7 cells. COUP-TFI also promotes the proliferation of MCF-7 cells through ER alpha-dependent mechanisms that target cell cycle progression and cell survival. To further investigate the mechanisms underlying these effects of COUP-TFI, we evaluated the expression of known E2-target genes in breast cancer, and found that COUP-TFI differentially regulated genes involved in cell proliferation, apoptosis, and migration/invasion. Notably, Cathepsin D (CTSD) transcript and protein levels were significantly higher in presence and absence of E2 in MCF-7 over-expressing COUP-TFI. Chromatin Immunoprecipitation assays showed that ER alpha, phospho-RNA Polymerase II, as well as p68 RNA Helicase, a phospho-Serine 118 dependent co-activator of ER alpha, were preferentially recruited onto the CTSD gene proximal promoter in COUP-TFI over-expressing cells. These results suggest that COUP-TFI selectively regulates the expression of endogenous E2-target genes and consequently modifies ER alpha positive mammary cells response to E2.

Walker G, MacLeod K, Williams AR, et al.
Estrogen-regulated gene expression predicts response to endocrine therapy in patients with ovarian cancer.
Gynecol Oncol. 2007; 106(3):461-8 [PubMed] Related Publications
OBJECTIVE: To explore the predictive value of estrogen-regulated gene changes as indicators of sensitivity in ovarian cancer patients treated with the aromatase inhibitor Letrozole.
METHODS: Expression of a range of proteins was assessed by semi-quantitative immunohistochemistry in tissue sections from the tumors of patients treated with Letrozole. Expression was correlated with clinical response to Letrozole. Corresponding mRNA in ovarian cancer cell lines treated with 17beta-estradiol (E2) was measured by quantitative RT-PCR.
RESULTS: In an estrogen receptor (ER)-positive ovarian cancer cell line, quantitative RT-PCR analysis demonstrated that PLAU, VIM, BIGH3, CDH6, FN1, CASP4, KRT4, KRT7, KRT13, TRAM and NGAL were down-regulated and TFF1, TFF3, TRAP1, TFAP4, MYC, CTSD, IL17BR, TOP2A, CCNB1, CCNB2, PDZK1 and UBE2C were up-regulated by E2. The E2 modulation of these genes was reversed by the anti-estrogen tamoxifen and was ERalpha-dependent. For ovarian cancer patients treated with Letrozole, we tested the predictive value of the majority of these genes in paraffin sections from their primary tumors by semi-quantitative immunohistochemistry. Significant differences in expression levels of TFF1, TFF3, BIGH3, TRAP1, VIM, TOP2A, PLAU and UBE2C were observed between tumors from CA125 responsive/stable patients as opposed to tumors from patients whose disease progressed, using serum levels of CA125 as an indicator of response. Aromatase expression in the ovarian cancers also differed between these 2 groups of patients.
CONCLUSION: These results suggest that expression levels of certain proteins in ovarian cancers are estrogen-regulated and could help identify patients who would benefit from endocrine therapy.

Bacac M, Provero P, Mayran N, et al.
A mouse stromal response to tumor invasion predicts prostate and breast cancer patient survival.
PLoS One. 2006; 1:e32 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
Primary and metastatic tumor growth induces host tissue responses that are believed to support tumor progression. Understanding the molecular changes within the tumor microenvironment during tumor progression may therefore be relevant not only for discovering potential therapeutic targets, but also for identifying putative molecular signatures that may improve tumor classification and predict clinical outcome. To selectively address stromal gene expression changes during cancer progression, we performed cDNA microarray analysis of laser-microdissected stromal cells derived from prostate intraepithelial neoplasia (PIN) and invasive cancer in a multistage model of prostate carcinogenesis. Human orthologs of genes identified in the stromal reaction to tumor progression in this mouse model were observed to be expressed in several human cancers, and to cluster prostate and breast cancer patients into groups with statistically different clinical outcomes. Univariate Cox analysis showed that overexpression of these genes is associated with shorter survival and recurrence-free periods. Taken together, our observations provide evidence that the expression signature of the stromal response to tumor invasion in a mouse tumor model can be used to probe human cancer, and to provide a powerful prognostic indicator for some of the most frequent human malignancies.

Yates CM, Patel A, Oakley K, et al.
Erythropoietin in thyroid cancer.
J Endocrinol Invest. 2006; 29(4):320-9 [PubMed] Related Publications
Erythropoietin (Epo) and the epo-receptor (EpoR) have been implicated in tumor growth, invasion and metastasis. We previously demonstrated Epo and EpoR expression in a small group of archived papillary thyroid cancers (PTC), but were unable to examine functional integrity using formalin-fixed tissues. In the present study, we examined the in vitro expression, induction and function of Epo and EpoR in papillary (NPA), follicular (WRO) and anaplastic (ARO-81) thyroid cancer cells. We found that all three cell lines expressed Epo and EpoR mRNA and that the hypoxia-mimetic cobalt induced Epo expression in all cell lines. None of the growth factors we examined (thyrotropin, vascular endothelial growth factor, IGF-I, or human Epo) altered Epo or EpoR gene expression. Importantly, however, administration of Epo to NPA but not WRO cells resulted in significant alterations in the expression of several mitogenic genes including cyclooxygenase-2 (COX-2), beta-casein (CSN2), wild type p53-induced gene-1 (WIG1) and cathepsin D (CTSD). Epo treated ARO-81 cells only had an increase in CSN2 expression. We conclude that Epo and EpoR are expressed by thyroid cancers and that stimulation of the Epo/EpoR signal pathway results in changes that could impact on the clinical behavior of thyroid cancers.

Fang WY, Liu TF, Xie WB, et al.
Reexploring the possible roles of some genes associated with nasopharyngeal carcinoma using microarray-based detection.
Acta Biochim Biophys Sin (Shanghai). 2005; 37(8):541-6 [PubMed] Related Publications
In gene expression profiling, nasopharyngeal carcinoma (NPC) 5-8F cells differ from 6-10B cells in terms of their high tumorigenicity and metastatic ability. Differentially expressed genes from the two cell types were analyzed by combining with MILANO (the automatic custom annotation of microarray results which is based on all the available published work in PubMed). The results showed that five genes, including CTSD, P63, CSE1L, BPAG1 and EGR1, have been studied or mentioned in published work on NPC. Subsequently, we reevaluated the roles of these genes in the pathogenesis of NPC by combining the data of gene chips from NPCs versus NPs and pooled cells from 5-8F, 6-10B and CNE2 versus NPs. The results suggested that the roles of BPAG1 and EGR1 are possibly different from those reported in previous NPC studies. These five genes are likely to be involved in the proliferation, apoptosis, invasion and metastasis of NPC. A reexploration of the genes will further define their roles in the pathogenesis of NPC.

Vendrell JA, Magnino F, Danis E, et al.
Estrogen regulation in human breast cancer cells of new downstream gene targets involved in estrogen metabolism, cell proliferation and cell transformation.
J Mol Endocrinol. 2004; 32(2):397-414 [PubMed] Related Publications
We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17beta-estradiol (E(2)) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. The specificity of the E(2) response was reinforced by comparison with 4-hydroxytamoxifen (OH-Tam), ICI 182,780 and E(2)+OH-Tam expression profiles. Real-time quantitative PCR (RTQ-PCR) confirmed the variation of expression of known (TFF1, AREG, IRS1, IGFBP4, PCNA, ERBB2, CTSD, MYC) as well as novel (DLEU2, CCNA2, UGT1A1, ABCC3, ABCC5, TACC1, EFNA1, NOV, CSTA, MMP15, ZNF217) genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E(2) treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E(2) on the transcriptional program of human E(2)-responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.

Stronach EA, Sellar GC, Blenkiron C, et al.
Identification of clinically relevant genes on chromosome 11 in a functional model of ovarian cancer tumor suppression.
Cancer Res. 2003; 63(24):8648-55 [PubMed] Related Publications
Microcell-mediated transfer of normal chromosome 11 (chr 11) to a clonal derivative of the ovarian cancer cell line, OVCAR3, was performed and generated independent hybrids with a common set of phenotypes: inhibition of cell growth and of cellular migration in vitro; and inhibition of tumor growth in vivo. Differential display reverse transcriptase-PCR (RT-PCR), cDNA-representational difference analysis, and hybridization of cDNA high-density filter arrays identified altered mRNAs associated with these phenotypic alterations. Quantitative RT-PCR-based validation of each altered mRNA eliminated false positives to identify a reduced set of expression differences. Twelve products were confirmed as up-regulated and 4 as down-regulated upon introduction of chr 11. Strikingly, 4 of the 12 up-regulated genes were located on chr 11. Expression analysis of selected products by quantitative RT-PCR in a series of 18 human primary ovarian tumors revealed several associations with clinicopathological features. Importantly, low expression of two products, the lysosomal protease CTSD and the lens crystallin CRYAB, was significantly associated with adverse patient survival. Immunohistochemical analysis of CTSD in a larger independent panel of 58 primary ovarian tumors confirmed that low CTSD was associated with poor survival. Furthermore, low CTSD was significantly associated with serous histology and advanced tumor stage. The combined approach of microcell-mediated chromosome transfer and expression difference analysis has identified several altered mRNAs in a model of chr 11-mediated ovarian tumor suppression. The detailed contextual characterization of these genes will determine the extent of their involvement in neoplastic development.

Atkins KB, Troen BR
Comparative responsiveness of HL-60, HL-60R, and HL-60R+ (LRARSN) cells to retinoic acid, calcitriol, 9 cis-retinoic acid, and sodium butyrate.
Blood. 1995; 86(7):2475-80 [PubMed] Related Publications
In HL-60 cells, retinoic acid (RA) and 9 cis-RA induce granulocytic differentiation, and calcitriol and sodium butyrate induce monocytic differentiation. To study the role of retinoid resistance on the response to these agents, we investigated their effects in HL-60 cells, retinoid-resistant HL-60R cells, and HL-60R+ cells in which retinoid sensitivity has been restored. In HL-60 cells, cathepsin D (ctsd) mRNA levels are increased by these agents and by cholera toxin after pretreatment with each agent. Calcitriol, 9 cis-RA, and sodium butyrate increase interleukin-8 (IL-8) mRNA expression, and pretreatment with these agents or RA potentiates the stimulation of IL-8 by phorbol ester (TPA). Pretreatment of HL-60 cells with all of the agents confers inducibility of cathepsin L (ctsl) mRNA by TPA in previously unresponsive cells. In HL-60R cells, none of the agents alone or in combination significantly enhances the expression of the ctsd, IL-8, or ctsl mRNAs. Retinoid stimulation (either alone or in combination with the other agents) of the three mRNAs is partially restored in the HL-60R+ cells. Calcitriol does not alter the expression of any of these mRNAs, and only the stimulation of IL-8 mRNA by sodium butyrate is recovered. Treatment with all of the agents inhibits proliferation and stimulates differentiation of the HL-60 cells. RA and calcitriol are unable to inhibit proliferation of the HL-60R cells, whereas only calcitriol fails to inhibit proliferation of the HL-60R+ cells. None of the agents induces differentiation in either the HL-60R or HL-60R+ cells. Therefore, the mutation of the RA receptor alpha is insufficient to account for the altered responses of the HL-60R cells, and there are likely defects in other signaling pathways in these cells. These cells may prove useful in examining the mechanism of cross-resistance between various differentiating agents.

Richard CW, Boehnke M, Berg DJ, et al.
A radiation hybrid map of the distal short arm of human chromosome 11, containing the Beckwith-Wiedemann and associated embryonal tumor disease loci.
Am J Hum Genet. 1993; 52(5):915-21 [PubMed] Article available free on PMC after 01/03/2015 Related Publications
We describe a high-resolution radiation hybrid (RH) map of the distal short arm of human chromosome 11 containing the Beckwith-Wiedemann gene and the associated embryonal tumor disease loci. Thirteen human 11p15 genes and 17 new anonymous probes were mapped by a statistical analysis of the cosegregation of markers in 102 rodent-human radiation hybrids retaining fragments of human chromosome 11. The 17 anonymous probes were generated from lambda phage containing human 11p15.5 inserts, by using ALU-PCR. A comprehensive map of all 30 loci and a framework map of nine clusters of loci ordered at odds of 1,000:1 were constructed by a multipoint maximum-likelihood approach by using the computer program RHMAP. This RH map localizes one new gene to chromosome 11p15 (WEE1), provides more precise order information for several 11p15 genes (CTSD, H19, HPX, ST5, RNH, and SMPD1), confirms previous map orders for other 11p15 genes (CALCA, PTH, HBBC, TH, HRAS, and DRD4), and maps 17 new anonymous probes within the 11p15.5 region. This RH map should prove useful in better defining the positions of the Beckwith-Wiedemann and associated embryonal tumor disease-gene loci.

Henry I, van Heyningen V, Puech A, et al.
Reassessment of breakpoints in chromosome 11p15.
Cytogenet Cell Genet. 1993; 62(1):52-3 [PubMed] Related Publications
Specific tumor-associated rearrangements involving the regions 11p13 and 11p15 have been extensively documented. However, cytogenetic definition of the breakpoints occurring at the boundaries of these two regions was not precise enough to correlate with the molecular data. Using probes corresponding to the genes coding for MYOD1, CTSD, LDHA, and RBTN1 and to the anonymous sequence D11S776, we have reassessed the breakpoints of three hybrids (J1.10, BID7, and NYX3.1) and confirmed the localization or more precisely mapped these four genes and the anonymous DNA marker on different subregions of 11pter-->p13, including the smallest region of 11p15.5 duplicated in a patient with Beckwith-Wiedemann syndrome.

Glaser T, Housman D, Lewis WH, et al.
A fine-structure deletion map of human chromosome 11p: analysis of J1 series hybrids.
Somat Cell Mol Genet. 1989; 15(6):477-501 [PubMed] Related Publications
Deletion analysis offers a powerful alternative to linkage and karyotypic approaches for human chromosome mapping. A panel of deletion hybrids has been derived by mutagenizing J1, a hamster cell line that stably retains chromosome 11 as its only human DNA, and selecting for loss of MIC1, a surface antigen encoded by a gene in band 11p13. A unique, self-consistent map was constructed by analyzing the pattern of marker segregation in 22 derivative cells lines; these carry overlapping deletions of 11p13, but selectively retain a segment near the 11p telomere. The map orders 35 breakpoints and 36 genetic markers, including 3 antigens, 2 isozymes, 12 cloned genes, and 19 anonymous DNA probes. The deletions span the entire short arm, dividing it into more than 20 segments and define a set of reagents that can be used to rapidly locate any newly identified marker on 11p, with greatest resolution in the region surrounding MIC1. The approach we demonstrate can be applied to map any mammalian chromosome. To test the gene order, we examined somatic cell hybrids from five patients, whose reciprocal translocations bisect band 11p13; these include two translocations associated with familial aniridia and two with acute T-cell leukemia. In each patient, the markers segregate in telomeric and centromeric groups as predicted by the deletion map. These data locate the aniridia gene (AN2) and a recurrent T-cell leukemia breakpoint (TCL2) in the marker sequence, on opposite sides of MIC1. To provide additional support, we have characterized the dosage of DNA markers in a patient with Beckwith-Wiedemann syndrome and an 11p15-11pter duplication. Our findings suggest the following gene order: TEL - (HRAS1, MER2, CTSD, TH/INS/IGF2, H19, D11S32) - (RRM1, D11S1, D11S25, D11S26) - D11S12 - (HBBC, D11S30) - D11S20 - (PTH, CALC) - (LDHA, SAA, TRPH, D11S18, D11S21) - D11S31 - D11S17 - HBVS1 - (FSHB, D11S16) - AN2 - MIC1 - TCL2 - delta J - CAT - MIC4 - D11S9 - D11S14 - ACP2 - (D11S33, 14L) - CEN. We have used the deletion map to show the distribution on 11p of two centromeric repetitive elements and the low-order interspersed repeat A36Fc. Finally, we provide evidence for an allelic segregation event in the hamster genome that underlies the stability of chromosome 11 in J1. The deletion map provides a basis to position hereditary disease loci on 11p, to distinguish the pattern of recessive mutations in different forms of cancer and, since many of these genes have been mapped in other mammalian species, to study the evolution of a conserved syntenic group.

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