Expression Of Prominin-1 Research Articles

Design, synthesis and biological evaluation of novel DCLK1 inhibitor containing purine skeleton for the treatment of pancreatic cancer

Pancreatic cancer is a highly lethal form of malignancy that continues to pose a significant and unresolved health challenge. Doublecortin-like kinase 1 (DCLK1), a serine/threonine kinase, is found to be overexpressed in pancreatic cancer and holds promise as a potential therapeutic target for this disease. However, few potent inhibitors have been reported currently. Herein, a series of novel purine, pyrrolo [2,3-d]pyrimidine, and pyrazolo [3,4-d] pyrimidine derivatives were designed, synthesized, and evaluated their biological activities in vitro. Among them, compound I-5 stood out as the most potent compound with strong inhibitory activity against DCLK1 (IC50 = 171.3 nM) and remarkable antiproliferative effects on SW1990 cell lines (IC50 = 0.6 μM). Notably, I-5 exhibited higher in vivo antitumor potency (Tumor growth inhibition value (TGI): 68.6 %) than DCLK1-IN-1 (TGI: 24.82 %) in the SW1990 xenograft model. The preliminary mechanism study demonstrated that I-5 not only inhibited SW1990 cell invasion and migration, but also decreased the expression of prominin-1 (CD133) and cluster of differentiation 44 (CD44), which are considered as differentiation markers for SW1990 stem cells. All the results indicated that I-5, a novel DCLK1 inhibitor, shows promise for further investigation in the treatment of pancreatic cancer.

Central role of Prominin-1 in lipid rafts during liver regeneration

Prominin-1, a lipid raft protein, is required for maintaining cancer stem cell properties in hepatocarcinoma cell lines, but its physiological roles in the liver have not been well studied. Here, we investigate the role of Prominin-1 in lipid rafts during liver regeneration and show that expression of Prominin-1 increases after 2/3 partial hepatectomy or CCl4 injection. Hepatocyte proliferation and liver regeneration are attenuated in liver-specific Prominin-1 knockout mice compared to wild-type mice. Detailed mechanistic studies reveal that Prominin-1 interacts with the interleukin-6 signal transducer glycoprotein 130, confining it to lipid rafts so that STAT3 signaling by IL-6 is effectively activated. The overexpression of the glycosylphosphatidylinsositol-anchored first extracellular domain of Prominin-1, which is the domain that binds to GP130, rescued the proliferation of hepatocytes and liver regeneration in liver-specific Prominin-1 knockout mice. In summary, Prominin-1 is upregulated in hepatocytes during liver regeneration where it recruits GP130 into lipid rafts and activates the IL6-GP130-STAT3 axis, suggesting that Prominin-1 might be a promising target for therapeutic applications in liver transplantation.

Hepatocyte-specific Prominin-1 protects against liver injury-induced fibrosis by stabilizing SMAD7

Prominin-1 (PROM1), also known as CD133, is expressed in hepatic progenitor cells (HPCs) and cholangiocytes of the fibrotic liver. In this study, we show that PROM1 is upregulated in the plasma membrane of fibrotic hepatocytes. Hepatocellular expression of PROM1 was also demonstrated in mice (Prom1CreER; R26TdTom) in which cells expressed TdTom under control of the Prom1 promoter. To understand the role of hepatocellular PROM1 in liver fibrosis, global and liver-specific Prom1-deficient mice were analyzed after bile duct ligation (BDL). BDL-induced liver fibrosis was aggravated with increased phosphorylation of SMAD2/3 and decreased levels of SMAD7 by global or liver-specific Prom1 deficiency but not by cholangiocyte-specific Prom1 deficiency. Indeed, PROM1 prevented SMURF2-induced SMAD7 ubiquitination and degradation by interfering with the molecular association of SMAD7 with SMURF2. We also demonstrated that hepatocyte-specific overexpression of SMAD7 ameliorated BDL-induced liver fibrosis in liver-specific Prom1-deficient mice. Thus, we conclude that PROM1 is necessary for the negative regulation of TGFβ signaling during liver fibrosis.

Prominin 1 Significantly Correlated with Bone Metastasis of Breast Cancer and Influenced the Patient's Prognosis.

Background This study is aimed at identifying the important biomarkers associated with bone metastasis (BM) in breast cancer (BRCA). Methods The GSE175692 dataset was used to detect significant differential expressed genes (DEGs) between BRCA samples with or without BM, and DEG-related pathways were then explored. Further, we constructed the protein-protein interaction (PPI) network on GEGs and filtered 5 vital nodes. We then performed the Cox regression, Kaplan-Meier analysis, nomogram, and ROC curve to filter the most significant prognosis genes. The GSE14020 and GSE124647 datasets were used to verify the expression and prognostic value of hub genes, respectively. Finally, the gene set enrichment analysis (GSEA) was performed to reveal the potential mechanism. Results Totally, 74 DEGs were detected, which mainly correlated with infectious disease, signaling molecules, and interaction. The 5 important DEGs were then filtered, and the Cox regression further showed that 2 genes, including prominin 1 (PROM1) and C-C motif chemokine ligand 2 (CCL2), were related to the prognosis of BRCA metastasis patients. Especially, PROM1 presented a better prognostic performance on the survival probability of patients than CCL2. Verification analysis further confirmed the abnormal expression and significant prognostic influence of PROM1. Finally, GSEA revealed that PROM1 was negatively related to IGF1 and mTOR pathways in BRCA metastasis. Conclusion PROM1 was an important biomarker associated with BRCA bone metastasis and affected the prognosis of metastatic BRCA patients. It may play a vital role in metastatic BRCA by negatively regulating IGF1 and mTOR pathways.

Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition.

Simple SummaryThe aim of this study was to gain a better understanding of cancer stem cells, which are a small subpopulation of tumor cells with high plasticity driving tumor growth and metastasis. Here we isolated two novel colorectal cancer cell lines originating from a rectal neuroendocrine carcinoma and a colorectal adenocarcinoma, depicting stem-like properties. These in vitro models offer the possibility to evaluate pathophysiological mechanisms in order to develop tailored therapeutic strategies for distinct colorectal malignancies. Investigations revealed gene copy number gain of the N-myc proto-oncogene for both. Accordingly, inhibition of the protein–protein interaction of myc and N-myc proto-oncogenes with the myc-associated factor X utilizing small molecule KJ-Pyr-9, exhibited a significant reduction in survival of both cell lines by the induction of apoptosis. Consequently, the blockage of these interactions may serve as a possible treatment strategy for colorectal cancer cell lines with gene copy number gain of the N-myc proto-oncogene.Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial–mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.

Napabucasin overcomes cisplatin resistance in ovarian germ cell tumor-derived cell line by inhibiting cancer stemness

BackgroundCisplatin resistance of ovarian yolk sac tumors (oYST) is a clinical challenge due to dismal patient prognosis, even though the disease is extremely rare. We investigated potential association between cisplatin resistance and cancer stem cell (CSC) markers in chemoresistant oYST cells and targeting strategies to overcome resistance in oYST.MethodsChemoresistant cells were derived from chemosensitive human oYST cells by cultivation in cisplatin in vitro. Derivative cells were characterized by chemoresistance, functional assays, flow cytometry, gene expression and protein arrays focused on CSC markers. RNAseq, methylation and microRNA profiling were performed. Quail chorioallantoic membranes (CAM) with implanted oYST cells were used to analyze the micro-tumor extent and interconnection with the CAM. Tumorigenicity in vivo was determined on immunodeficient mouse model. Chemoresistant cells were treated by inhibitors intefering with the CSC properties to examine the chemosensitization to cisplatin.ResultsLong-term cisplatin exposure resulted in seven-fold higher IC50 value in resistant cells, cross-resistance to oxaliplatin and carboplatin, and increased migratory capacity, invasiveness and tumorigenicity, associated with hypomethylation of differentially methylated genes/promotors. Resistant cells exhibited increased expression of prominin-1 (CD133), ATP binding cassette subfamily G member 2 (ABCG2), aldehyde dehydrogenase 3 isoform A1 (ALDH3A1), correlating with reduced gene and promoter methylation, as well as increased expression of ALDH1A3 and higher overall ALDH enzymatic activity, rendering them cross-resistant to DEAB, disulfiram and napabucasin. Salinomycin and tunicamycin were significantly more toxic to resistant cells. Pretreatment with napabucasin resensitized the cells to cisplatin and reduced their tumorigenicity in vivo.ConclusionsThe novel chemoresistant cells represent unique model of refractory oYST. CSC markers are associated with cisplatin resistance being possible targets in chemorefractory oYST.

T140 Inhibits Apoptosis and Promotes Proliferation and Matrix Formation Through the SDF-1/CXC Receptor-4 Signaling Pathway in Endplate Chondrocytes of the Rat Intervertebral Discs

Cartilaginous endplate (CEP), a thin layer of hyaline cartilage located between the vertebral endplate and nucleus pulposus, transports the nutrient into the disc. The objective of this study was to evaluate the influence of T140 (polyphemusin II-derived peptide) on the CEP cell growth, apoptosis, and the matrix formation via the stromal cell-derived factor-1 (SDF-1)/cysteine X cysteine (CXC) receptor-4 (CXCR4) signaling pathway. Sprague-Dawley rats were euthanized by cervical dislocation and dissected for the isolation and the appraisal of CEP cells that were extracted from the endplate in rat intervertebral discs and were then added with different concentrations of reagents (SDF-1 and T140). The effect of T140 on CEP cell proliferation and apoptosis were analyzed. The messenger RNA (mRNA) and protein expressions of CXCR4, prominin-1, proteoglycans, type II collagen, B-cell lymphoma-2 (Bcl-2), and Bcl-2 associated X protein were analyzed by reverse transcription quantitative polymerase chain reaction and Western blot analysis. T140 promoted the proliferation of CEP cells and inhibited the apoptosis of CEP cells. Additionally, T140 suppressed the mRNA and protein expression of CXCR4, prominin-1, and Bcl-2 associated X protein, and increased the mRNA and protein expression of proteoglycans, type II collagen, and Bcl-2. T140 promotes the proliferation and matrix formation and inhibits the apoptosis of CEP cells by blocking the SDF-1/CXCR4 signaling pathway invitro, which provides a certain therapeutic effect on the degeneration of intervertebral discs.

Prominin-1 (CD133) modulates the architecture and dynamics of microvilli.

Prominin-1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non-epithelial cells. Irrespective of the cell type, prominin-1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin-Darby canine kidney cells as the main model. Our high-resolution analysis revealed that upon the overexpression of prominin-1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob-like morphologies were observed and stimulated by mutations in the ganglioside-binding site of prominin-1. The altered phenotypes were caused by the interaction of prominin-1 with phosphoinositide 3-kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin-1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin-1-ganglioside membrane complexes, phosphoinositide 3-kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin-1 and its mutants modified the structure of filopodia emerging from fibroblast-like cells and silencing human prominin-1 in primary hematopoietic stem cells resulted in the loss of uropod-associated microvilli. Altogether, these findings strengthen the role of prominin-1 as an organizer of cellular protrusions.

Hepatic Prominin-1 expression is associated with biliary fibrosis

Intrahepatic biliary fibrosis, as seen with cholestatic liver injuries such as biliary atresia, is mechanistically distinct from fibrosis caused by hepatocyte toxicity. We previously demonstrated the expansion of cells expressing the stem/progenitor cell marker Prominin-1, within regions of developing fibrosis in biliary atresia. Thus, we hypothesized that Prominin-1 expression is biliary fibrosis-specific. Gene expression of Prominin-1 was analyzed in adult mice undergoing either cholestatic bile duct ligation or hepatotoxic carbon tetrachloride administration by quantitative polymerase chair reaction. Lineage tracing of Prominin-1-expressing cells and Collagen-1α-expressing cells was performed after bile duct ligation in Prominin-1cre-ert2-lacz;Gfplsl and Collagen-1αGfp transgenic mice, respectively. Prominin-1 expression increased significantly after bile duct ligation compared with sham (6.6±0.9-fold change at 2weeks, P<.05) but not with carbon tetrachloride (-0.7±0.5-fold change, not significant). Upregulation of Prominin-1 was observed histologically throughout the liver as early as 5days after bile duct ligation in Prominin-1cre-ert2-lacz mice by LacZ staining in nonhepatocyte cells. Lineage tracing of Prominin-1-expressing cells labeled prior to bile duct ligation in Prominin-1cre-ert2-lacz;Gfplsl mice, demonstrated increasing colocalization of GREEN FLUORESCENT PROTEIN with biliary marker CYTOKERATIN-19 within ductular reactions up to 5weeks after bile duct ligation consistent with biliary transdifferentiation. In contrast, rare colocalization of GREEN FLUORESCENT PROTEIN with mesenchymal marker α-SMOOTH MUSCLE ACTIN in Prominin-1cre-ert2-lacz;Gfplsl mice and some colocalization of GREEN FLUORESCENT PROTEIN with PROMININ-1 in Collagen-1αGfp mice, indicate minimal contribution of Prominin-1 progenitor cells to the pool of collagen-producing myofibroblasts. During biliary fibrosis Prominin-1-expressing progenitor cells transdifferentiate into cells within ductular reactions. This transdifferentiation may promote fibrosis.

Immunological and Clinical Impact of Cancer Stem Cells in Vulvar Cancer: Role of CD133/CD24/ABCG2-Expressing Cells.

Cancer stem cells (CSCs) are tumour-initiating cells with self-renewal properties and chemo/radio-resistance. Regulatory T-cells (Tregs) influence CSCs through several mechanisms. In different solid tumours, the presence of both cell populations correlated with poor survival. In vulvar cancer, little is known regarding biological markers able to predict patient prognosis. We investigated the presence and clinical impact of CSCs and infiltrating Treg in primary vulvar cancer. Paraffin-embedded tissue specimens derived from 43 patients with vulvar cancer were analyzed by immunohistochemistry for the expression of prominin-1 (CD133), CD24, ATP-binding cassette sub-family G member 2 (ABCG2) (CSC markers) and forkhead box protein P3 (FOXP3) (Treg marker). CD133 expression correlated with younger age at diagnosis (p<0.01), lymph-node metastasis (p<0.05) and larger tumour diameter (p<0.05). CD133+ tumours showed a high FOXP3+ T-cell infiltration. Overall survival and progression-free survival were not influenced by the expression of the analyzed biomarkers. In vulvar cancer, CSCs were more frequently expressed in younger aged patients and those with aggressive disease. Their presence was also associated with high Treg infiltration, which contributes to the generation of an immunosuppressive milieu.

High CD133 expression in the nucleus and cytoplasm predicts poor prognosis in non-small cell lung cancer.

Objective. The aim of this study was to investigate the expression of Prominin-1 (CD133) in cancer cells and its potential value as a prognostic indicator of survival in patients with non-small cell lung cancer (NSCLC). Methods. Cancerous tissues and matched normal tissues adjacent to the carcinoma from 239 NSCLC patients were obtained immediately after surgery. Immunohistochemistry of tissue microarrays was used to characterize the expression of CD133 in NSCLC and adjacent tissues. The correlation of CD133 expression with clinical characteristics and prognosis was determined by statistical analysis. Results. CD133 protein expression levels in both the cytoplasm and nucleus were significantly higher in NSCLC tissues compared with corresponding peritumoral tissue (P < 0.05). CD133 expression in the nucleus of NSCLC cells was related to tumor diameter (P = 0.027), tumor differentiation (P < 0.001), and TNM stage (P = 0.007). Kaplan-Meier survival and Cox regression analyses revealed that high CD133 expression in the nucleus was an independent predictor of poor prognosis of NSCLC, as was high cytoplasmic CD133 expression (P < 0.001). Conclusion. Our findings provide the first evidence that high expression of CD133 in both the nucleus and cytoplasm is associated with poor prognosis in NSCLC.

Spatial Distribution of Prominin-1 (CD133) – Positive Cells within Germinative Zones of the Vertebrate Brain

BackgroundIn mammals, embryonic neural progenitors as well as adult neural stem cells can be prospectively isolated based on the cell surface expression of prominin-1 (CD133), a plasma membrane glycoprotein. In contrast, characterization of neural progenitors in non-mammalian vertebrates endowed with significant constitutive neurogenesis and inherent self-repair ability is hampered by the lack of suitable cell surface markers. Here, we have investigated whether prominin-1–orthologues of the major non-mammalian vertebrate model organisms show any degree of conservation as for their association with neurogenic geminative zones within the central nervous system (CNS) as they do in mammals or associated with activated neural progenitors during provoked neurogenesis in the regenerating CNS.MethodsWe have recently identified prominin-1 orthologues from zebrafish, axolotl and chicken. The spatial distribution of prominin-1–positive cells – in comparison to those of mice – was mapped in the intact brain in these organisms by non-radioactive in situ hybridization combined with detection of proliferating neural progenitors, marked either by proliferating cell nuclear antigen or 5-bromo-deoxyuridine. Furthermore, distribution of prominin-1 transcripts was investigated in the regenerating spinal cord of injured axolotl.ResultsRemarkably, a conserved association of prominin-1 with germinative zones of the CNS was uncovered as manifested in a significant co-localization with cell proliferation markers during normal constitutive neurogenesis in all species investigated. Moreover, an enhanced expression of prominin-1 became evident associated with provoked, compensatory neurogenesis during the epimorphic regeneration of the axolotl spinal cord. Interestingly, significant prominin-1–expressing cell populations were also detected at distinct extraventricular (parenchymal) locations in the CNS of all vertebrate species being suggestive of further, non-neurogenic neural function(s).Conclusion/InterpretationCollectively, our work provides the first data set describing a comparative analysis of prominin-1–positive progenitor cells across species establishing a framework for further functional characterization in the context of regeneration.

Expression and localisation of osteopontin and prominin-1 (CD133) in patients with endometriosis

In this study, we investigated the expression and localisation of the proteins, osteopontin (OPN) and prominin-1 (CD133), as well as the plasma OPN levels in the endometrium of patients with endometriosis. Samples of ectopic endometriotic lesions and normal endometrium were obtained from 31 women with endometriosis and 28 healthy control subjects. The mRNA and protein expression of OPN and CD133 was analysed by real-time RT-PCR and immunohistochemistry. The plasma levels of OPN were determined by ELISA. Our results revealed that OPN mRNA and protein expression, as well as its release in the blood, was significantly increased in the endometriotic lesions in comparison to normal tissue. Although the presence of CD133+ cells was detected in the normal endometrium, as well as in the endometriosis specimens, a significant quantitative variation of this protein was not demonstrated in the patients with endometriosis. In conclusion, our data indicate that OPN is involved in the development of endometriosis by enhancing the invasiveness, proliferation and survival of endometrial cells in ectopic lesions. CD133 cannot be used as a disease marker for endometriosis, although an involvement of this protein in the pathogenesis of endometriosis cannot be excluded.

Prominin-1 Allows Prospective Isolation of Neural Stem Cells from the Adult Murine Hippocampus

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However, despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone, no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo, including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting, only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation, self-renewal, and multipotentiality as assessed by a neurosphere assay. In addition, isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally, despite its expression by various stem and progenitor cells, Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless, a net decrease in hippocampal neurogenesis, by ∼30% was found in Prominin-1 knock-out mice, suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably, an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism, which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene.

Prominin-1 and Its Role in Tumor Progression and Assessment of Clinical Prognosis in Systemic Malignancies

To the Editor: The recent article by Di Bonito et al. [1] provided for highly stimulating reading. Prominin-1 may influence tumor progression and clinical prognosis in a number of other systemic tumors. A similar relationship is seen in colorectal cancers. In fact, prominin-1 exerts significant influence on tumor progression in colonic malignancies. Nearly, 36% of all malignant colonic masses are positive for prominin-1 expression [2]. Decreased “overall survival” is seen in patients with up-regulated prominin-1 levels. Accentuated prominin-1 expression is associated with accentuated tumor invasiveness. In general, “microsatellite instability-high” tumors express lower levels of prominin-1 in contrast to “microsatellite stable” colorectal tumors [3]. Not surprisingly, only 9% of colorectal tumors with a good prognosis are positive for prominin-1. In contrast, nearly 52% of colorectal malignancies that exhibit a poor clinical outcome are positive for prominin-1. Lymph node metastasis is also more prominent in tumors positive for prominin-1. Interestingly, the expression of prominin-1 by malignant colonic tissue is decreased following preoperative treatment with nonsteroidal anti-inflammatory agents [4]. Prominin-1 expression also plays a role in tumor progression in prostatic malignancies. For instance, tumor cells that are positive for prominin-1 expression tend to proliferate at a significantly more accentuated rate in comparison to prom inin-1 negative cancer cells [5,6]. Not surprisingly, tumor cells in the G-2 phase are primarily composed of prominin-1 positive cells. A similar association is seen in ovarian malignancies. Nearly 31% of all ovarian cancers are positive for prominin-1 expression. A poor clinical prognosis is seen in patients with ovarian tumors with accentuated prominin-1 expression as is reflected in the shorter “disease-free survival” times noted in these patients [7]. The above examples illustrate the significant role that prom inin-1 plays in tumor progression and its potential use as a significant prognostic maker.

High expression of octamer-binding transcription factor 4A, prominin-1 and aldehyde dehydrogenase strongly indicates involvement in the initiation of lung adenocarcinoma resulting in shorter disease-free intervals

The increasing relevance of the cancer stem cell (CSC) hypothesis and the impact of CSC-associated markers in the carcinogenesis of solid tumours may provide potential prognostic implications in lung cancer. We propose that a collective genetic analysis of established CSC-related markers will generate data to better define the role of putative CSCs in lung adenocarcinoma (LAC). Sixty-four paired tumour and non-tumour biopsies from LAC patients were included in this study. Using the quantitative reverse transcriptase-polymerase chain reaction, we assessed the expression profiles of established CSC-related biomarkers: octamer-binding transcription factor 4 (OCT4A), CD133, aldehyde dehydrogenase (ALDH), BMI-1, ATP-binding cassette subfamily G, member 2 (ABCG2), SRY (sex-determining region Y)-box 2 (SOX2) and uPAR, and evaluated their relation to clinicopathological parameters and disease prognosis. All of the above-mentioned CSC-related markers were detectable in both tumour and corresponding normal tissues. Importantly, expression levels of OCT4A, CD133, BMI-1, SOX2 and uPAR were significantly higher (OCT4A, P = 0.0003; CD133, P = 0.002; BMI-1, P = 0.04; SOX2, P = 0.0003; uPAR, P = 0.03) in the tumour compared with those in the non-tumour tissues. By contrast, the quantities of ACBG2 and ALDH were markedly reduced (ACBG2, P = 0.0006; ALDH, P = 0.007) in the tumour relative to those in the normal biopsies. Using multivariate analysis, elevated ALDH and CD133 revealed significant associations in tumour stage (ALDH, P = 0.03; CD133, P = 0.007) and differentiation (ALDH, P = 0.03; CD133, P = 0.018). We observed that ALDH and OCT4A were associated with nodal status (ALDH, P = 0.05; OCT4A, P = 0.03) having lower mRNA levels in tumours with lymph node metastasis, N+, compared with that in N0. High OCT4A levels were significantly correlated with tumour size of <3 cm, decrease in tumours >3 cm (P = 0.03). Kaplan-Meier correlation analyses, showed that OCT4A and CD133 were correlated to short disease-free intervals (OCT4A, P = 0.047; CD133, P = 0.033) over a period of 29 months. Our study reveals that CSC-associated markers: OCT4A, CD133 and ALDH are involved in the initial phase of carcinogenesis of LAC, and can be used as predictors of early stage LAC and poor disease-free intervals. In addition, this work validates the relevance of the CSC hypothesis in LAC.

Combination of a Ptgs2 Inhibitor and an Epidermal Growth Factor Receptor-Signaling Inhibitor Prevents Tumorigenesis of Oligodendrocyte Lineage-Derived Glioma-Initiating Cells

Recent findings have demonstrated that malignant tumors, including glioblastoma multiforme (GBM), contain cancer-initiating cells (CICs; also known as cancer stem cells), which self-renew and are malignant. However, it remains controversial whether such CICs arise from tissue-specific stem cells, committed precursor cells, or differentiated cells. Here, we sought to examine the origin of the CICs in GBM. We first showed that the overexpression of oncogenic HRas(L61) transformed p53-deficient oligodendrocyte precursor cells (OPCs) and neural stem cells (NSCs) into glioma-initiating cell (GIC)-like cells in mice. When as few as 10 of these GIC-like cells were transplanted in vivo, they formed a transplantable GBM with features of human GBM, suggesting that these GIC-like cells were enriched in CICs. DNA microarray analysis showed that widespread genetic reprogramming occurred during the OPCs' transformation: they largely lost their OPC characteristics and acquired NSC ones, including the expression of prominin1, hmga2, ptgs2, and epiregulin. In addition, the combination of a Ptgs2 inhibitor and an epidermal growth factor receptor (EGFR)-signaling inhibitor prevented the tumorigenesis of transformed OPCs and human GICs (hGICs) obtained from anaplastic oligodendroglioma, but not of transformed NSCs or hGICs obtained from GBM. Together, these findings suggest that GBM can arise from either OPCs or NSCs and that the therapeutic targets for GBM might be different, depending on each GIC's cell-of-origin.

Distinct and Conserved Prominin-1/CD133–Positive Retinal Cell Populations Identified across Species

Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133) plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts—except for the axolotl—were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells), but they also marked distinct subdivisions of the inner nuclear layer (INL). In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b), a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6–positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1–positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1–positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1–expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

Expression and localization of prominin-1 in isoproterenol-treated rat parotid gland

Prominin-1 (CD133) is a pentaspan cholesterol-binding membrane glycoprotein. Chronic treatment with isoproterenol, a beta-receptor agonist, induces several dramatic effects on salivary glands, such as enhanced DNA synthesis and proliferation of salivary acinar cells. In addition, the biosynthetic pathways of membrane lipids may be altered by the isoproterenol stimulation. The purpose of this study was to investigate the effect of isoproterenol administration on prominin-1 expression profiles in rat parotid gland by means of PCR and immunohistochemistry. Rats were chronically treated with the beta-adrenergic agonist for 1, 3, and 7 days. Our results showed that isoproterenol-treatment caused a down-regulation of prominin-1 on day 3 and 7 of treatment, as well as a differential immunostaining distribution pattern. This study suggests that isoproterenol-treatment may represent a useful tool to explore the molecular mechanisms involved in the synthesis and release of prominin-1. Such efforts could contribute to the development of diagnostic tools based on the detection of prominin-1 in biological fluids, such as saliva.

Abstract 4640: High expression of the stem cell marker LGR5 is associated with a poor clinical outcome in patients with Ewing's sarcoma

Abstract Ewing's sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that are most commonly diagnosed in childhood and adolescence. Unfortunately, drug-resistant relapse is not uncommon and is associated with very poor outcomes. No clinical or pathologic features currently exist that reliably predict chemosensitivity or relapse in newly diagnosed patients. In this study we used whole genome expression microarray profiling to identify potential markers of drug resistance and relapse in ESFT. Affymetrix human exon (HuEx) arrays were used to profile a highly drug resistant case of primary, localized ESFT before and after induction therapy. Comparison of gene summarized data from pre- and post-therapy samples with 10 chemosensitive tumors identified 309 genes to be differentially expressed in the resistant case at diagnosis (fold change&amp;gt;2). Of the 309 differentially expressed genes at diagnosis, 130 demonstrated an even greater change in expression post-induction therapy suggesting that these genes might be markers of the most highly drug-resistant cells. Intriguingly, two stem cell associated genes, PROM1 (prominin 1) and LGR5 (leucine-rich-repeat containing G-protein-coupled receptor 5), were included in the list of putative chemoresistance markers. Expression of PROM1 and LGR5 were 18-fold and 17-fold higher, respectively, at diagnosis and 31-fold and 21-fold higher post-induction therapy in the drug resistant case. To determine if either gene is associated with chemoresistant disease or relapse in ESFT we next evaluated whether their expression correlated with outcome in a large cohort of institutional and Children's Oncology Group patients. Clinical correlates analysis of HuEx gene expression data obtained from 53 tumors revealed no correlation between PROM1 expression and outcome. In contrast, increased expression of LGR5 was associated with worse event-free (p=0.008) and overall survival (p=0.046). Variable but detectable expression of LGR5 in ESFT tumors and cell lines was validated using quantitative RT-PCR. Finally, LGR5 expression in ESFT cells was compared to human embryonic and somatic stem cells and fibroblasts. Significantly, LGR5 was found to be highly expressed by neural crest stem cells but not by bone-marrow derived mesenchymal stem cells, differentiated neural crest cells, or embryonic fibroblasts. These data suggest that LGR5 might be useful as a predictor of outcome in ESFT and implicate a primitive neural crest stem cell phenotype in the developmental origins of drug-resistant and relapsed disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4640.