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Lgr5 expression, cancer stem cells and pancreatic cancer: results from biological and computational analyses Kalliopi Andrikou1, Matteo Santoni1, Francesco Piva2, Alessandro Bittoni1, Andrea Lanese1, Chiara Pellei1, Alessandro Conti3, Cristian Loretelli1, Alessandra Mandolesi4, Matteo Giulietti2, Marina Scarpelli4, Giovanni Principato2, Massimo Falconi5 & Stefano Cascinu*,1
ABSTRACT Aims: To determine the relationship between Lgr5 and other stemness markers and pathologic features in pancreatic ductal adenocarcinoma (PDAC) samples. Materials & methods: In 69 samples, Lgr5 was analyzed by qRT-PCR together with a panel of 29 genes. Bioinformatic analysis was carried out to identify a possible pathway regulating Lgr5 expression in PDAC. Results: Lgr5 expression was not associated with the expression of tested cancer stem cell markers. Moreover, it was not an independent predictor of survival neither at univariate analysis (p = 0.21) nor at multivariate analysis (p = 0.225). Conclusion: Based on the lack of correlation between Lgr5 and tested cancer stem cell markers, Lgr5 does not seem to be a potential stemness marker or prognostic factor in PDAC.
Pancreatic cancer is one of the most lethal human malignancies, with an overall 5-year survival rate of no more than 5% and a median overall survival of around 6 months [1] . Such a dismal prognosis is the consequence not only of a late diagnosis but also of an intrinsic resistance to chemotherapeutic drugs due to specific tumor features. Therefore, a better understanding of disease biology and identification of new biomarkers may lead to the development of more effective treatment strategies. Cancer stem cells (CSCs) represent a distinct population of tumor cells, functionally defined by their ability to self-renew and to differentiate [2,3] . The activation of embryonic signaling pathways, such as the Wnt, Notch and Hedgehog, has been suggested to drive the formation of CSCs [4] . The CSC hypothesis is a topic of great interest, since it seems to be the basis for the phenomenon of chemotherapy resistance. However, the role of CSC in pancreatic ductal adenocarcinoma (PDAC) remains controversial. In fact, despite the importance of CD44, CD133 and ALDH in identifying pancreatic CSCs, it is still unclear whether these antigens merely serve as phenotypic markers or are involved in regulating CSC function [5] . Other pancreatic CSC markers have been identified, such as SDF-1 receptor or CXCR4, which is expressed by a subset of CD133 + CSCs with enhanced metastatic capacity [2] . The cell surface receptor Lgr5, also known as GPR49, is a receptor for the secreted glycoproteins RSPO1, an activator of Wnt signaling [6,7] . Lgr5 promotes the growth and survival of different cancer cell types, including colorectal and basal cell carcinoma through an enhancement of canonical
KEYWORDS
• cancer stem cell • embryonic signaling pathways • Lgr5 • overall survival • pancreatic ductal
adenocarcinoma
Medical Oncology, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy 2 Department of Clinical Sciences, Polytechnic University of the Marche Region, Piazza Roma 22, 60121 Ancona, Italy 3 Section of Urology, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy 4 Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy 5 Pancreatic Surgery Unit, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy *Author for correspondence: Tel.: +39 071 596 4169; Fax: +39 071 596 4192; [email protected] 1
10.2217/FON.15.27 © 2015 Future Medicine Ltd
Future Oncol. (2015) 11(7), 1037–1045
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Research Article Andrikou, Santoni, Piva et al. Wnt/β-catenin signaling [8] . These results suggest a possible link between Lgr5, Wnt signaling, CSCs and cancer progression [9] . Furthermore, retrospective studies have shown that high levels of Lgr5 are associated with worse survival in colorectal cancer and glioblastoma [10,11] , suggesting a possible prognostic role. In pancreatic cancer, little is known about the role of Lgr5. The aim of this study is to analyze Lgr5 expression in human PDAC tissue samples exploring its relationship with other CSC ma rkers and pathological features. Materials & methods ●●Patient characteristics
Tissue samples as well as clinical data from 69 pancreatic cancer patients, non-metastatic at diagnosis, who underwent surgical resection were collected between 2006 and 2012. All patients provided written consent to have their samples and linked clinical data banked for clinical and basic research purposes. Our institutional ethical committee approved the consent process. For this study, all samples and data were de-identified and, therefore, did not require any additional institutional review board approval. Clinical and pathological characteristics of patients were retrieved from the institutional database. ●●Samples processing & quantitative PCR
analysis
Gene expression profile analysis was performed by laboratory personnel blinded to patients’ status. The tissue samples were fixed in 10% neutral-buffered formalin for 14–24 h within 1 h after surgical removal, before being alcoholdehydrated and embedded in paraffin. Formalinfixed, paraffin-embedded tissue samples were cut in 10-μm-thick sections on a microtome with a disposable blade. Total RNA from pancreatic cancer specimen was extracted from formalin-fixed, paraffin-embedded tissue using RT2 FFPE RNA Extraction Kit (SABiosciences Corporation, MD, USA) after tissue sections had been deparaffinized using the method with Proteinase K digestion and rehydrated in 100% ethanol and 75% ethanol according to the supplier’s instructions. After isolation, RNA samples were treated with Ambion® TURBO DNA-free™ kit for minimizing or eliminating genomic DNA contamination. RNA samples were quantified by a NanoDrop® spectrophotometric assay. Five hundred nanograms from each sample were reverse transcribed
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to cDNA and pre-amplified using the RT2 FFPE PreAMP cDNA Synthesis Kit and the primer mix specific for the customized Stem Cell RT2Profiler PCR Array (SABiosciences Corporation). Quantitative real-time PCR analysis was performed on a 7300 real-time PCR System (Applied Biosystems, Inc., CA, USA) by a SYBRH Green method. ●●Data processing & statistical analysis
Relative gene expression was quantified using the comparative ΔCt method. We used the tool ‘PCR Array Data Analysis Web Portal’ [12] on the manufacturer’s website to perform data quality tests, calculations on the qPCR data and data normalization. In particular, all threshold cycles values greater than 35 or not detected were considered as negative calls. We retained samples with negative genomic DNA control, definite reverse transcription control and positive PCR control values, according to manufacturer’s indications. Lgr5 expression levels were normalized to that of three housekeeping genes: HPRT1, B2M and GUSB. The survival time was defined as the duration from the date of surgery to the date of death. Cases of death from other causes or lost to follow-up were treated as censored cases. Survival analysis was conducted via Kaplan–Meier product-limit method and the Mantel–Haenszel log-rank test was employed to compare survival among groups. The association between the expression level of Lgr5 and the other 28 genes (Table 1) was performed by the Pearson product moment correlation method (rho 0.7 → strong correlation). The assumption of proportionality of hazards was checked with graphic analysis of scaled Schoenfeld residuals. Variables not fitting at univariate analysis were excluded from the multivariate model. No multicollinearity of the grouped co-variates was checked. Significance level in the univariate model for inclusion in the multivariate final model was more liberally set at a 0.2 level, according to Hosmer et al. [13,14] . All other significance levels were set at a 0.05 value and all p-values were two-sided. We consider to be able to detect an intermediate effect size (0.3, as proposed by Cohen), alpha = 0.05, with the sample size collected of 69 patients. No assumptions are made on the direction of the correlation (positive or negative), so a two-sided test is
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Lgr5 expression, cancer stem cells & pancreatic cancer
Research Article
Table 1. Genes analyzed, main gene function and correlation with Lgr5 expression by Pearson product moment correlation method. Gene ID
Gene name
Biological process
Correlation with Lgr5 expression
ALCAM B2M† BMP4 BRCA1 BRCA2 CD24‡ CD44‡ CDKN2A DHH FLT1 GUSB† HGF HPRT1† IHH Lgr5
Cell adhesion Housekeeping gene for normalization Cell differentiation Cell cycle Cell cycle Cell proliferation Cell adhesion Cell cycle regulation and apoptosis Cell differentiation Cell differentiation and angiogenesis Housekeeping gene for normalization Cell proliferation, chemotaxis and apoptosis Housekeeping gene for normalization Cell differentiation Cell development, stem cell marker
-0.109 0.110 0.29 -0.11 0.133 -0.19 0.057 0.278 0.155 -0.138 0.49 1
MET
CD166 antigen Beta-2-microglobulin Bone morphogenetic protein 4 Breast cancer type 1 susceptibility protein Breast cancer type 2 susceptibility protein Signal transducer CD24 CD44 antigen Cyclin-dependent kinase inhibitor 2A, isoform 4 Desert hedgehog protein Vascular endothelial growth factor receptor 1 Beta-glucuronidase Hepatocyte growth factor Hypoxanthine-guanine phosphoribosyltransferase Indian hedgehog protein Leucine-rich repeat-containing G-protein coupled receptor 5 Hepatocyte growth factor receptor
-0.074
MUC6 NOTCH1 OCT3/4 PDGFRB PGF PROM1 PTCH1 PTCH2 SHH SMAD4 SMO SPARC VEGFA VEGFB VEGFR-2 WNT1
Mucin-6 Neurogenic locus notch homolog protein 1 POU domain, class 5, transcription factor 1 Platelet-derived growth factor receptor beta Placenta growth factor Prominin-1 Protein patched homolog 1 Protein patched homolog 2 Sonic hedgehog protein Mothers against decapentaplegic homolog 4 Smoothened homolog Secreted protein acidic and rich in cysteine Vascular endothelial growth factor A Vascular endothelial growth factor B Vascular endothelial growth factor receptor 2 Proto-oncogene Wnt-1
Cell proliferation, scattering, morphogenesis and survival Maintenance of epithelium Cell differentiation and angiogenesis Cell differentiation Cell proliferation, chemotaxis and migration Cell differentiation and angiogenesis Cell differentiation Cell differentiation Cell differentiation Cell differentiation Cell differentiation and signal transduction Cell differentiation Cell growth Cell differentiation and angiogenesis Cell differentiation and angiogenesis Cell differentiation and angiogenesis Cell differentiation
0.050 0.128 0.159 -0.04 0.175 0.304 0.118 -0.05 0.575 0.012 0.111 0.020 -0.02 0.108 -0.115 0.0052
Housekeeping genes. Stem cell markers.
† ‡
chosen. Under these conditions, achieved statistical power for our correlations is 75%. Statistical analysis was conducted with the ‘R’ statistical software version 2.15.2. ●●Bioinformatic analyses
Microarray expression data derived from Gene Expression Omnibus (GEO) [15,16] . We have considered GDS4102 [17] and GDS4103 [18] (both Affymetrix Human Genome U133 Plus 2.0 Array platform), GDS596 (Affymetrix Human Genome U133A Array platform) [19] and GDS4336 (Affymetrix Human Gene 1.0 ST
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Array platform) [20] datasets taking in account that experiments derived from different platforms, some expression data were logarithmic and there is more than one probe for some genes. Moreover the last dataset has been normalized by Robust Multi-array Average (RMA) method. Results ●●Patient characteristics
A total of 69 patients were included in this analysis. Median age was 71 years (range: 48−88 years). A total of 37 patients were male (54%). Patient characteristics are summarized in (Table 2) .
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Research Article Andrikou, Santoni, Piva et al. Table 2. Patient characteristics. Characteristics
Patients, n (%)
Patients Gender: – Male – Female Age (range), years TNM status: – T1 – T2 – T3 Node status: – N0 – N1 Grading: – Grade 1 – Grade 2 – Grade 3 Type of pancreactectomy performed: – Pancreaticoduodenectomy – Distal pancreatectomy – Total pancreatectomy
69 (100) 37 (54) 32 (46) 70 (47–87) 2 (3) 4 (6) 63 (91) 21 (30) 48 (70) 3 (4) 25 (36) 41 (60) 48 (69) 14 (20) 7 (11)
●●Analysis of gene expression & association
between Lgr5 & clinico-pathological features
Lgr5 expression was not associated with the expression of stem cell markers, such as CD24 (rho = 0.133), CD44 (rho = -0.19), OCT3/4 (rho = 0.159) and PROM1 (rho = 0.304). The list of analyzed genes and the correlation with Lgr5 expression by Pearson method are reported in Table 1. Moreover, Lgr5 expression levels were not associated with any of the pathological characteristics of PDAC. Clinical and pathological features analyzed included gender (male vs female), age (70 years), pT stage (pT1–pT2 vs pT3), pN stage (pN0 vs pN1), grading (G1–G2 vs G3) and Lgr5 RNA levels (more vs less than prespecified cut-off). Prespecified cut-off for Lgr5 was defined as the median expression level normalized for housekeeping genes. Overall median survival was 13.7 months (95% CI: 10.8−18.7 months). At univariate analysis Lgr5 expression was not an independent predictor of survival (p = 0.21) and also after adjusting for clinical-pathological features (patients age and tumor size), no significant improvement were observed (p = 0.225). Any other clinical parameter did not meet the significance level for inclusion in multivariate
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analysis. Results of univariate and multivariate regression are summarized in Table 3. ●●The regulation of Lgr5 expression: results
from computational analysis
Transcriptomic and proteomic databases to compare our Lgr5 expression data (0.44-fold in comparison with the mean of our house keeping-selected genes) with those reposed in public resources were analyzed. With regard to GDS4103 microarray experiments collected in GEO section of National Center for Biotechnology Information (NCBI), we have calculated Lgr5 expression value of 0.1-fold in comparison with the mean of our HK-selected genes, both in normal and in cancer samples. Instead GDS4102 dataset points out 0.0025fold for normal and 0.0083-fold for cancer, GDS4336 gives 0.44-fold for normal and 0.51fold for cancer, dataset GDS596 reports 0.02fold in normal samples but it does not store tumor samples. In addition, according to microarray data of RefExA (Reference database for gene Expression Analysis of the University of Tokyo [21]) Lgr5 is not expressed in normal pancreas, and according to SAGE data of CGAP (Cancer Genome Anatomy Project) [22] Lgr5 is nearly absent both in normal (5 tags) and tumor (6 tags) pancreatic tissues. According to Human Protein Atlas [23,24] , in normal pancreas Lgr5 protein is expressed on average in exocrine glandular cells and highly in Islets of Langerhans. Moreover, it is expressed at an intermediate level in 60%, low in 15% and absent in the remaining 15% of pancreatic cancer samples collected in the same web repository. A mass spectrometry experiment (identifier HuPA_00784) provides the insight that protein is present in normal pancreas in Human Proteinpedia [25,26] , while this is not reported in Human Protein Reference Database [27,28] and GenAtlas [29,30] . By analyzing literature data [31,32] , it is possible to define and to draw pathways where Lgr5 may be involved. Figure 1 shows that Wnt signaling promotes Lgr5 protein expression by increasing Lgr5 gene transcription. In turn Wnt, by increasing β-catenin, could increase c-Myc transcription, resulting in a decrease of miR-23 transcription and an increase of AP4 transcription leading to an increase of Lgr5 protein expression. Furthermore, c-Myc may decrease miR-34 transcription with an increase
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Lgr5 expression, cancer stem cells & pancreatic cancer of ZNF281 and then of Lgr5. Finally, ZNF281 acts also as negative feedback since it decreases β-catenin transcription. Discussion In the last decade, increasing attention has been focused on CSCs as potential therapeutic target in several human malignancies, including PDAC. However, the identification and the characterization of CSCs remain a challenge so far. Lgr5, a Wnt target gene, has been proposed as a CSC marker in colorectal and gastric cancers [33,34] . Fujishita et al. [35] showed that Wnt/ β-catenin signaling pathway is important for the activation of the mTOR pathway during intestinal polyp formation. Moreover, Ko et al. [36] proposed Lgr5 as a potential stem cell marker in pancreatic neuroendocrine tumors. Wnt signaling pathway plays a crucial role in the initiation and progression of PDAC [37,38] . Based on this evidence, Lgr5 expression has also been investigated in pancreatic cancer. Simon et al. [39] showed higher levels of Lgr5 expression, mainly cytoplasmic, in 17 PDAC samples compared with normal pancreatic tissue. Similarly, Mizuno et al. [40] showed that Lgr5 is expressed on the cytoplasm of pancreatic cancer cells and on the basolateral membrane of endocrine pancreatic cells in patients affected by PDAC. In our study, the expression of Lgr5 in tumor samples from patients with PDAC was investigated. As previously reported, we showed that PDAC cells express Lgr5. The present results were compared with previous data on normal pancreatic tissue and we found GDS4102 dataset showing that Lgr5 is overexpressed in tumor samples. The correlation between Lgr5 expression and other pancreatic CSC markers, such as CD24, CD44, OCT3/4 and PROM1 was also evaluated. Differently from other tumors,
Research Article
a significant correlation between LGR5 and the other CSC markers was not observed. Furthermore, several studies revealed a prognostic role of Lgr5 expression in a series of solid tumors, including colorectal [10,41–42] and gastric carcinoma [43] , glioblastoma [11] , esophageal adenocarcinoma [44] and lung cancer [45] . In addition, Lgr5 expression levels have been shown to be correlated with the clinical characteristics of cancer patients, such as tumor node metastasis stage, tumor grade, lymph node metastasis and vascular invasion [11,39,44,46] . In our study, Lgr5 expression was not significantly associated with the pathological characteristics as well as with the clinical outcome of PDAC patients. Based on these results, we investigated for the biological mechanisms underlying the role of Lgr5 in PDAC. Lgr5 gene yields three alternative splicing forms (wild-type LGR5/GPR49, GPR49Δ5 and VSP_037746) characterized by altered binding affinity to their ligands [47] . Rot el al. [48] described a splice variant of Lgr5 in the context of soft tissue sarcoma and reported that low expression levels of this variant transcript (which lacks exon 5) were associated with worse overall survival. Based on this observation, further studies are needed to assess the role of different Lgr5 splicing variants in PDAC. Furthermore, the contribution of Lgr5 to cancer maintenance and progression is likely to be determined by both genetic and epigenetic state of the affected cell as well as the surrounding microenvironment. Lgr5 has been suggested to be a target gene of miR-23b [41] , a miRNA commonly expressed in pancreatic cell lines [49] . Similarly, Lgr5 seems to be also the target gene of miR-142-3p, which has been shown to inhibit the Lgr5 protein expression in colon cancer [50] . Moreover, other miRNAs could regulate Lgr5, such as miR-216a, expressed in normal pancreas [51] but downregulated in PDAC [52] or miR-342-3p overexpressed in pancreatic
Table 3. Results of univariate and multivariate regression of Lgr5 expression level and of the pathological characteristics of pancreatic ductal adenocarcinoma. Variable Gender (male vs female) Age (70 years) pT (pT1–pT2 vs pT3) pN (pN0 vs pN1) Grading (grade 1–2 vs grade 3) LGR5 expression
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Univariate
Multivariate
Hazard ratio (95% CI) p-value
Hazard ratio (95% CI) p-value
1.24 (0.73–2.12) 1.92 (1.11–3.31) 2.02 (0.72–5.62) 1.25 (0.68–2.28) 1.17 (0.74–1.85) 1.33 (0.85–2.09)
1.73 (0.97–3.09) 1.56 (0.54–4.53) 1.31 (0.85–2.03)
0.424 0.019 0.180 0.475 0.512 0.21
0.065 0.416 0.225
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SLUG
c-Myc
AP4
miR-23
AP4
miR-23
ZEB1
ZEB2
TWIST
c-Myc
p53
E-cadherin
miR-34 ZNF281
LGR5
SNAIL miR-34
p21 LGR5
NOTCH1
SNAIL
ZNF281
SOX4
NOTCH1 β-catenin
β-catenin
Axin2
Degradation and phosphorylation
Gastrin ODCD
Axin2 CK1α GSK3 APC
Nanog
EMT
Wnt CD44
C-Myc
TCF-1
C-jun
MMP7
CD133
Cyclin D1
TCF4
Figure 1. Pathway in pancreatic cancer cells that shows the complex regulation of Lgr5 by Wnt signaling pathway. Wnt signaling promotes Lgr5 protein expression: by increasing Lgr5 gene transcription; by increasing c-Myc transcription, resulting in a decrease of miR-23 transcription and an increase of AP4 transcription; by decreasing miR-34 transcription with an increase of ZNF281 and then of Lgr5. The rectangles and double helix stylization indicate a gene and its promoter, while smoothed rectangles indicate a protein or a transcription factor. Alone rectangles indicate mature miRNA.
tumors [53,54] . However, the mechanisms by which miRNAs induce translational repression are still not clear and deserve ulterior investigations in PDAC. The main limitations of this study include the lack of data on Lgr5 protein levels, which could not directly reflect the RNA levels due to the post-transcriptional regulation, as well as its retrospective design, which is susceptible to bias in data selection and analysis. Moreover, other markers of stemness, such as CXCR4 or Nanog, have not been evaluated in this study.
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Despite these limitations, our study shows that Lgr5 does not seem to be correlated with the pathological characteristics and outcome of patients with PDAC, as well as with the expression of other evaluated CSCs markers. Lgr5 seems to be overexpressed in PDAC, but its role remains still controversial. Conclusion Our analyses suggest that Lgr5 RNA expression in human pancreatic tumor samples does not have a prognostic role in this disease. Moreover,
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Lgr5 expression, cancer stem cells & pancreatic cancer considering the lack of association between RNA expression levels of Lgr5 and of tested pancreatic CSC markers, Lgr5 does not seem to have a role as potential CSCs marker in PDAC. Future perspective Despite the development of new therapeutic approaches, pancreatic cancer remains one of the deadliest cancer-related diseases in the world [55] . Several groups demonstrated that CSCs are responsible for the resistance of this disease to conventional treatment [56,57] . Therefore, this group of undifferentiated cells seems to be a very intriguing target for therapy and actually findings suggest that specific elimination of these cells is possible and therapeutically relevant. Indeed, recently it has been demonstrated that a multimodal treatment including two relevant CSC pathway inhibitors and addition chemotherapy represents a very promising approach with a marked survival benefit in preclinical model [58] . Whereas further studies are needed to confirm these results, a depletion of CSCs may well become a powerful target in clinical cancer therapy. Furthermore, it is important to find markers that exclusively identify pancreatic CSCs but
Research Article
Lgr5 expression does not seems to be relevant in this field. Thus, further investigation is needed to evaluate the clinical significance of these findings and an accurate study to define specific pancreatic CSC markers that may lead to the development of novel treatment regimens in this disease. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
EXECUTIVE SUMMARY ●●
Wnt signaling pathway plays a crucial role in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC).
●●
Lgr5, a Wnt target gene, has been proposed as a potential stem cell marker in various human malignancies, but its role in PDAC remains still unclear.
●●
In 69 PDAC patients underwent surgical resection, Lgr5 seems to be overexpressed in tumor samples in comparison with normal tissue.
●●
Lgr5 RNA expression was not associated with the expression of stem cell markers, such as CD24 (rho = 0.133), CD44 (rho = -0.19), OCT3/4 (rho = 0.159) and PROM1 (rho = 0.304).
●●
Lgr5 RNA expression levels were not associated with any of the pathological characteristics of PDAC.
●●
At univariate analysis Lgr5 RNA expression was not an independent predictor of survival (p = 0.21).
●●
Further studies are needed to assess the role of different Lgr5 splicing variants in PDAC.
●●
Thus, currently Lgr5 does not seem to represent a potential cancer stem cell marker in PDAC. determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1(3), 313–323 (2007).
4
Takebe N, Ivy SP. Controversies in cancer stem cells: targeting embryonic signaling pathways. Clin. Cancer Res. 16(12), 3106–3112 (2010).
•
Shows that a subpopulation of migrating CD133 + CXCR4 + cancer stem cells is essential for tumor metastasis in pancreatic ductal adenocarcinoma.
5
Penchev VR, Rasheed ZA, Maitra A, Matsui W. Heterogeneity and targeting of pancreatic cancer stem cells. Clin. Cancer Res. 18(16), 4277–4284 (2012).
3
Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell 124(6), 1111–1115 (2006).
•• This review evaluated the potential relationships between different pancreatic
References Papers of special note have been highlighted as: • of interest; •• of considerable interest 1
Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J. Clin. 63(1), 11–30 (2013).
2
Hermann PC, Huber SL, Herrler T et al. Distinct populations of cancer stem cells
future science group
www.futuremedicine.com
1043
Research Article Andrikou, Santoni, Piva et al. cancer stem cell populations and strategies to identify novel targeting approaches. 6
7
8
9
Hao HX, Xie Y, Zhang Y et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485(7397), 195–200 (2012). Koo BK, Spit M, Jordens I et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488(7413), 665–669 (2012). Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc. Natl Acad. Sci. USA 108(28), 11452–11457 (2011). Clevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell 149(6), 1192–1205 (2012).
10 Wu XS, Xi HQ, Chen L. Lgr5 is a potential
marker of colorectal carcinoma stem cells that correlates with patient survival. World J. Surg. Oncol. 10, 244 (2012).
specifically overexpressed in tumor epithelia. Hepatogastroenterology 55(88), 2016–2027 (2008). 19 Su AI, Wiltshire T, Batalov S et al. A gene
atlas of the mouse and human proteinencoding transcriptomes. Proc. Natl Acad. Sci. USA 101(16), 6062–6067 (2004). 20 Zhang G, Schetter A, He P et al. DPEP1
inhibits tumor cell invasiveness, enhances chemosensitivity and predicts clinical outcome in pancreatic ductal adenocarcinoma. PLoS ONE 7(2), e31507 (2012). 21 Laboratory for System Biology and Medicine.
www.lsbm.org 22 Strausberg RL1, Buetow KH, Emmert-Buck
MR, Klausner RD. The cancer genome anatomy project: building an annotated gene index. Trends Genet. 16(3), 103–106 (2000). 23 Uhlen M, Oksvold P, Fagerberg L et al.
Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28, 1248–1250 (2010).
•• In patients with colorectal cancer, Lgr5 correlates with poor prognosis and may also be considered as a potential marker for colorectal cancer stem cells.
24 Protein Atlas.
11 Nakata S, Campos B, Bageritz J et al. LGR5 is
25 Kandasamy K, Keerthikumar S, Goel R et al.
a marker of poor prognosis in glioblastoma and is required for survival of brain cancer stem-like cells. Brain Pathol. 23(1), 60–72 (2013). •
Shows that LGR5 plays an important role in maintenance and/or survival of brain CSCs in this disease.
12 SABiosciences.
www.SABiosciences.com 13 Fagerland MW, Hosmer DW, Bofin AM.
Multinomial goodness-of-fit tests for logistic regression models. Stat. Med. 27(21), 4238–4253 (2008). 14 Mickey RM, Greenland S. The impact of
confounder selection criteria on effect estimation. Am. J. Epidemiol. 129(1), 125–137 (1989). 15 Edgar R, Domrachev M, Lash AE. Gene
Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30(1), 207–210 (2002).
Human Proteinpedia: a unified discovery resource for proteomics research. Nucleic Acids Res. 37, D773–D781 (2009). 26 Human Proteinpedia.
www.humanproteinpedia.org 27 Peri S, Navarro JD, Amanchy R et al.
Development of human protein reference database as an initial platform for approaching systems biology in humans. Genome Res. 13(10), 2363–2371 (2003). 28 Human Protein Reference Database.
www.hprd.org 29 Frézal J. Genatlas database, genes and
development defects. C. R. Acad. Sci. III 321(10), 805–817 (1998). 30 Gene Atlas.
www.genatlas.org 31 Jackstadt R, Roh S, Neumann J et al. AP4 is a
mediator of epithelial-mesenchymal transition and metastasis in colorectal cancer. J. Exp. Med. 210(7), 1331–1350 (2013). 32 Hahn S, Jackstadt R, Siemens H, Hunten S,
16 NCBI.
www.ncbi.nlm.nih.gov/geo/ 17 Pei H, Li L, Fridley BL, Jenkins GD et al.
FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt. Cancer Cell 16(3), 259–266 (2009). 18 Badea L, Herlea V, Dima SO, Dumitrascu T,
Popescu I. Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes
1044
www.proteinatlas.org
Hermeking H. SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial-mesenchymal transition. EMBO J. 32(23), 3079–3095 (2013). 33 Kember K, Prasetyanti PR, De Lau W,
Rodermond H, Clevers H, Medema JP. Monoclonal antibodies against Lgr5 identify human colorectal cancer stem cells. Stem Cells 30(11), 2378–2386 (2012).
Future Oncol. (2015) 11(7)
34 Zheng ZX, Sun Y, Bu ZD et al. Intestinal
stem cell marker LGR5 expression during gastric carcinogenesis. World J. Gastroenterol. 19(46), 8714–8721 (2013). 35 Fujishita T, Aoki K, Lane HA, Aoki M,
Taketo MM. Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in ApcΔ716 mice. Proc. Natl Acad. Sci. USA 105(36), 13544–13549 (2008). 36 Ko SB, Azuma S, Yokoyama Y et al.
Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas. Am. J Physiol. Gastrointest. Liver Physiol. 304(12), G1103–G1116 (2013). 37 Pasca di Magliano M, Biankin AV, Heiser
PW et al. Common activation of canonical Wnt signaling in pancreatic adenocarcinoma. PLoS ONE 2(11), e1155 (2007). 38 Morris JP, Cano DA, Sekine S, Wang SC,
Hebrok M. Beta-catenin blocks Krasdependent reprogramming of acini into pancreatic cancer precursor lesions in mice. J. Clin. Invest. 120(2), 508–520 (2010). 39 Simon E, Petke D, Boger C et al. The spatial
distribution of LGR5+ cells correlates with gastric cancer progression. PLoS ONE 7(4), e35486 (2012). 40 Mizuno N, Yatabe Y, Hara K et al.
Cytoplasmic expression of LGR5 in pancreatic adenocarcinoma. Front. Physiol. 4, 269 (2013). •• Demonstrated that LGR5 is expressed in the cytoplasm of pancreatic cancer cells and the basolateral membrane of endocrine cells of the pancreas in patients with PDA. 41 Saigusa S, Inoue Y, Tanaka K et al. Significant
correlation between LKB1 and LGR5 gene expression and the association with poor recurrence-free survival in rectal cancer after preoperative chemoradiotherapy. J. Cancer Res. Clin. Oncol. 139(1), 131–138 (2012). 42 Takahashi H, Ishii H, Nishida N et al.
Significance of Lgr5 (+ve) cancer stem cells in the colon and rectum. Ann. Surg. Oncol. 18(4), 1166–1174 (2011). 43 Zheng ZX, Sun Y, Bu ZD et al. Intestinal
stem cell marker LGR5 expression during gastric carcinogenesis. World J. Gastroenterol. 19(46), 8714–8721 (2013). 44 Becker L, Huang Q, Mashimo H. Lgr5, an
intestinal stem cell marker, is abnormally expressed in Barrett’s esophagus and esophageal adenocarcinoma. Dis. Esophagus. 23(2), 168–174 (2010). 45 Ryuge S, Sato Y, Jiang SX et al. The
clinicopathological significance of Lgr5 expression in lung adenocarcinoma. Lung Cancer 82(1), 143–148 (2013).
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Lgr5 expression, cancer stem cells & pancreatic cancer 46 Xi HQ, Cai AZ, Wu XS et al. Leucine-rich
repeat-containing G-protein-coupled receptor 5 is associated with invasion, metastasis, and could be a potential therapeutic target in human gastric cancer. Br. J. Cancer 110(8), 2011–2020 (2014). •
In this subset of patients, Lgr5 seems to be correlated with invasion and metastasis.
47 Chen PH, Chen X, Lin Z, Fang D, He X. The
structural basis of R-spondin recognition by LGR5 and RNF43. Genes Dev. 27(12), 1345–1350 (2013). 48 Rot S, Taubert H, Bache M et al. A novel
splice variant of the stem cell marker LGR5/ GPR49 is correlated with the risk of tumor-related death in soft-tissue sarcoma patients. BMC Cancer 11, 429 (2011). 49 Wang P, Zhang J, Zhang L et al. MicroRNA
23b regulates autophagy associated with radioresistance of pancreatic cancer cells. Gastroenterology 145(5), 1133–1143 (2013).
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50 Shen WW, Zeng Z, Zhu WX, Fu GH.
MiR-142-3p functions as a tumor suppressor by targeting CD133, ABCG2, and Lgr5 in colon cancer cells. J. Mol. Med. (Berl). 91(8), 989–1000 (2013). 51 Endo K, Weng H, Kito N, Fukushima Y, Iwai
N. MiR-216a and miR-216b as markers for acute phased pancreatic injury. Biomed. Res. 34(4), 179–188 (2013). 52 Szafranska AE, Davison TS, John J et al.
MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene 26(30), 4442–4452 (2007). 53 Roldo C, Missiaglia E, Hagan JP et al.
MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. J. Clin. Oncol. 24(29), 4677–4684 (2006).
Research Article
54 Lee YS, Dutta A. MicroRNAs in cancer.
Annu. Rev. Pathol. 4, 199–227 (2009). 55 Philip PA, Mooney M, Jaffe D et al.
Consensus report of the national cancer institute clinical trials planning meeting on pancreas cancer treatment. J. Clin. Oncol. 27, 5660–5669 (2009). 56 Hong SP, Wen J, Bang S, Park S, Song SY.
CD44-positive cells are responsible for gemcitabine resistance in pancreatic cancer cells. Int. J. Cancer 125(10), 2323–2331 (2009). 57 Jimeno A, Feldmann G, Suarez-Gauthier A
et al. A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development. Mol. Cancer Ther. 9, 2582–2592 (2009). 58 Mueller MT, Hermann PC, Witthauer J et al.
Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer. Gastroenterology 137, 1102–1113 (2009).
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Lgr5 expression, cancer stem cells and pancreatic cancer: results from biological and computational analyses Kalliopi Andrikou1, Matteo Santoni1, Francesco Piva2, Alessandro Bittoni1, Andrea Lanese1, Chiara Pellei1, Alessandro Conti3, Cristian Loretelli1, Alessandra Mandolesi4, Matteo Giulietti2, Marina Scarpelli4, Giovanni Principato2, Massimo Falconi5 & Stefano Cascinu*,1
ABSTRACT Aims: To determine the relationship between Lgr5 and other stemness markers and pathologic features in pancreatic ductal adenocarcinoma (PDAC) samples. Materials & methods: In 69 samples, Lgr5 was analyzed by qRT-PCR together with a panel of 29 genes. Bioinformatic analysis was carried out to identify a possible pathway regulating Lgr5 expression in PDAC. Results: Lgr5 expression was not associated with the expression of tested cancer stem cell markers. Moreover, it was not an independent predictor of survival neither at univariate analysis (p = 0.21) nor at multivariate analysis (p = 0.225). Conclusion: Based on the lack of correlation between Lgr5 and tested cancer stem cell markers, Lgr5 does not seem to be a potential stemness marker or prognostic factor in PDAC.
Pancreatic cancer is one of the most lethal human malignancies, with an overall 5-year survival rate of no more than 5% and a median overall survival of around 6 months [1] . Such a dismal prognosis is the consequence not only of a late diagnosis but also of an intrinsic resistance to chemotherapeutic drugs due to specific tumor features. Therefore, a better understanding of disease biology and identification of new biomarkers may lead to the development of more effective treatment strategies. Cancer stem cells (CSCs) represent a distinct population of tumor cells, functionally defined by their ability to self-renew and to differentiate [2,3] . The activation of embryonic signaling pathways, such as the Wnt, Notch and Hedgehog, has been suggested to drive the formation of CSCs [4] . The CSC hypothesis is a topic of great interest, since it seems to be the basis for the phenomenon of chemotherapy resistance. However, the role of CSC in pancreatic ductal adenocarcinoma (PDAC) remains controversial. In fact, despite the importance of CD44, CD133 and ALDH in identifying pancreatic CSCs, it is still unclear whether these antigens merely serve as phenotypic markers or are involved in regulating CSC function [5] . Other pancreatic CSC markers have been identified, such as SDF-1 receptor or CXCR4, which is expressed by a subset of CD133 + CSCs with enhanced metastatic capacity [2] . The cell surface receptor Lgr5, also known as GPR49, is a receptor for the secreted glycoproteins RSPO1, an activator of Wnt signaling [6,7] . Lgr5 promotes the growth and survival of different cancer cell types, including colorectal and basal cell carcinoma through an enhancement of canonical
KEYWORDS
• cancer stem cell • embryonic signaling pathways • Lgr5 • overall survival • pancreatic ductal
adenocarcinoma
Medical Oncology, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy 2 Department of Clinical Sciences, Polytechnic University of the Marche Region, Piazza Roma 22, 60121 Ancona, Italy 3 Section of Urology, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy 4 Section of Pathological Anatomy, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy 5 Pancreatic Surgery Unit, Polytechnic University of the Marche Region, School of Medicine, United Hospitals, Via Conca 71, 60126 Ancona, Italy *Author for correspondence: Tel.: +39 071 596 4169; Fax: +39 071 596 4192; [email protected] 1
10.2217/FON.15.27 © 2015 Future Medicine Ltd
Future Oncol. (2015) 11(7), 1037–1045
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Research Article Andrikou, Santoni, Piva et al. Wnt/β-catenin signaling [8] . These results suggest a possible link between Lgr5, Wnt signaling, CSCs and cancer progression [9] . Furthermore, retrospective studies have shown that high levels of Lgr5 are associated with worse survival in colorectal cancer and glioblastoma [10,11] , suggesting a possible prognostic role. In pancreatic cancer, little is known about the role of Lgr5. The aim of this study is to analyze Lgr5 expression in human PDAC tissue samples exploring its relationship with other CSC ma rkers and pathological features. Materials & methods ●●Patient characteristics
Tissue samples as well as clinical data from 69 pancreatic cancer patients, non-metastatic at diagnosis, who underwent surgical resection were collected between 2006 and 2012. All patients provided written consent to have their samples and linked clinical data banked for clinical and basic research purposes. Our institutional ethical committee approved the consent process. For this study, all samples and data were de-identified and, therefore, did not require any additional institutional review board approval. Clinical and pathological characteristics of patients were retrieved from the institutional database. ●●Samples processing & quantitative PCR
analysis
Gene expression profile analysis was performed by laboratory personnel blinded to patients’ status. The tissue samples were fixed in 10% neutral-buffered formalin for 14–24 h within 1 h after surgical removal, before being alcoholdehydrated and embedded in paraffin. Formalinfixed, paraffin-embedded tissue samples were cut in 10-μm-thick sections on a microtome with a disposable blade. Total RNA from pancreatic cancer specimen was extracted from formalin-fixed, paraffin-embedded tissue using RT2 FFPE RNA Extraction Kit (SABiosciences Corporation, MD, USA) after tissue sections had been deparaffinized using the method with Proteinase K digestion and rehydrated in 100% ethanol and 75% ethanol according to the supplier’s instructions. After isolation, RNA samples were treated with Ambion® TURBO DNA-free™ kit for minimizing or eliminating genomic DNA contamination. RNA samples were quantified by a NanoDrop® spectrophotometric assay. Five hundred nanograms from each sample were reverse transcribed
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to cDNA and pre-amplified using the RT2 FFPE PreAMP cDNA Synthesis Kit and the primer mix specific for the customized Stem Cell RT2Profiler PCR Array (SABiosciences Corporation). Quantitative real-time PCR analysis was performed on a 7300 real-time PCR System (Applied Biosystems, Inc., CA, USA) by a SYBRH Green method. ●●Data processing & statistical analysis
Relative gene expression was quantified using the comparative ΔCt method. We used the tool ‘PCR Array Data Analysis Web Portal’ [12] on the manufacturer’s website to perform data quality tests, calculations on the qPCR data and data normalization. In particular, all threshold cycles values greater than 35 or not detected were considered as negative calls. We retained samples with negative genomic DNA control, definite reverse transcription control and positive PCR control values, according to manufacturer’s indications. Lgr5 expression levels were normalized to that of three housekeeping genes: HPRT1, B2M and GUSB. The survival time was defined as the duration from the date of surgery to the date of death. Cases of death from other causes or lost to follow-up were treated as censored cases. Survival analysis was conducted via Kaplan–Meier product-limit method and the Mantel–Haenszel log-rank test was employed to compare survival among groups. The association between the expression level of Lgr5 and the other 28 genes (Table 1) was performed by the Pearson product moment correlation method (rho 0.7 → strong correlation). The assumption of proportionality of hazards was checked with graphic analysis of scaled Schoenfeld residuals. Variables not fitting at univariate analysis were excluded from the multivariate model. No multicollinearity of the grouped co-variates was checked. Significance level in the univariate model for inclusion in the multivariate final model was more liberally set at a 0.2 level, according to Hosmer et al. [13,14] . All other significance levels were set at a 0.05 value and all p-values were two-sided. We consider to be able to detect an intermediate effect size (0.3, as proposed by Cohen), alpha = 0.05, with the sample size collected of 69 patients. No assumptions are made on the direction of the correlation (positive or negative), so a two-sided test is
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Lgr5 expression, cancer stem cells & pancreatic cancer
Research Article
Table 1. Genes analyzed, main gene function and correlation with Lgr5 expression by Pearson product moment correlation method. Gene ID
Gene name
Biological process
Correlation with Lgr5 expression
ALCAM B2M† BMP4 BRCA1 BRCA2 CD24‡ CD44‡ CDKN2A DHH FLT1 GUSB† HGF HPRT1† IHH Lgr5
Cell adhesion Housekeeping gene for normalization Cell differentiation Cell cycle Cell cycle Cell proliferation Cell adhesion Cell cycle regulation and apoptosis Cell differentiation Cell differentiation and angiogenesis Housekeeping gene for normalization Cell proliferation, chemotaxis and apoptosis Housekeeping gene for normalization Cell differentiation Cell development, stem cell marker
-0.109 0.110 0.29 -0.11 0.133 -0.19 0.057 0.278 0.155 -0.138 0.49 1
MET
CD166 antigen Beta-2-microglobulin Bone morphogenetic protein 4 Breast cancer type 1 susceptibility protein Breast cancer type 2 susceptibility protein Signal transducer CD24 CD44 antigen Cyclin-dependent kinase inhibitor 2A, isoform 4 Desert hedgehog protein Vascular endothelial growth factor receptor 1 Beta-glucuronidase Hepatocyte growth factor Hypoxanthine-guanine phosphoribosyltransferase Indian hedgehog protein Leucine-rich repeat-containing G-protein coupled receptor 5 Hepatocyte growth factor receptor
-0.074
MUC6 NOTCH1 OCT3/4 PDGFRB PGF PROM1 PTCH1 PTCH2 SHH SMAD4 SMO SPARC VEGFA VEGFB VEGFR-2 WNT1
Mucin-6 Neurogenic locus notch homolog protein 1 POU domain, class 5, transcription factor 1 Platelet-derived growth factor receptor beta Placenta growth factor Prominin-1 Protein patched homolog 1 Protein patched homolog 2 Sonic hedgehog protein Mothers against decapentaplegic homolog 4 Smoothened homolog Secreted protein acidic and rich in cysteine Vascular endothelial growth factor A Vascular endothelial growth factor B Vascular endothelial growth factor receptor 2 Proto-oncogene Wnt-1
Cell proliferation, scattering, morphogenesis and survival Maintenance of epithelium Cell differentiation and angiogenesis Cell differentiation Cell proliferation, chemotaxis and migration Cell differentiation and angiogenesis Cell differentiation Cell differentiation Cell differentiation Cell differentiation Cell differentiation and signal transduction Cell differentiation Cell growth Cell differentiation and angiogenesis Cell differentiation and angiogenesis Cell differentiation and angiogenesis Cell differentiation
0.050 0.128 0.159 -0.04 0.175 0.304 0.118 -0.05 0.575 0.012 0.111 0.020 -0.02 0.108 -0.115 0.0052
Housekeeping genes. Stem cell markers.
† ‡
chosen. Under these conditions, achieved statistical power for our correlations is 75%. Statistical analysis was conducted with the ‘R’ statistical software version 2.15.2. ●●Bioinformatic analyses
Microarray expression data derived from Gene Expression Omnibus (GEO) [15,16] . We have considered GDS4102 [17] and GDS4103 [18] (both Affymetrix Human Genome U133 Plus 2.0 Array platform), GDS596 (Affymetrix Human Genome U133A Array platform) [19] and GDS4336 (Affymetrix Human Gene 1.0 ST
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Array platform) [20] datasets taking in account that experiments derived from different platforms, some expression data were logarithmic and there is more than one probe for some genes. Moreover the last dataset has been normalized by Robust Multi-array Average (RMA) method. Results ●●Patient characteristics
A total of 69 patients were included in this analysis. Median age was 71 years (range: 48−88 years). A total of 37 patients were male (54%). Patient characteristics are summarized in (Table 2) .
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Research Article Andrikou, Santoni, Piva et al. Table 2. Patient characteristics. Characteristics
Patients, n (%)
Patients Gender: – Male – Female Age (range), years TNM status: – T1 – T2 – T3 Node status: – N0 – N1 Grading: – Grade 1 – Grade 2 – Grade 3 Type of pancreactectomy performed: – Pancreaticoduodenectomy – Distal pancreatectomy – Total pancreatectomy
69 (100) 37 (54) 32 (46) 70 (47–87) 2 (3) 4 (6) 63 (91) 21 (30) 48 (70) 3 (4) 25 (36) 41 (60) 48 (69) 14 (20) 7 (11)
●●Analysis of gene expression & association
between Lgr5 & clinico-pathological features
Lgr5 expression was not associated with the expression of stem cell markers, such as CD24 (rho = 0.133), CD44 (rho = -0.19), OCT3/4 (rho = 0.159) and PROM1 (rho = 0.304). The list of analyzed genes and the correlation with Lgr5 expression by Pearson method are reported in Table 1. Moreover, Lgr5 expression levels were not associated with any of the pathological characteristics of PDAC. Clinical and pathological features analyzed included gender (male vs female), age (70 years), pT stage (pT1–pT2 vs pT3), pN stage (pN0 vs pN1), grading (G1–G2 vs G3) and Lgr5 RNA levels (more vs less than prespecified cut-off). Prespecified cut-off for Lgr5 was defined as the median expression level normalized for housekeeping genes. Overall median survival was 13.7 months (95% CI: 10.8−18.7 months). At univariate analysis Lgr5 expression was not an independent predictor of survival (p = 0.21) and also after adjusting for clinical-pathological features (patients age and tumor size), no significant improvement were observed (p = 0.225). Any other clinical parameter did not meet the significance level for inclusion in multivariate
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analysis. Results of univariate and multivariate regression are summarized in Table 3. ●●The regulation of Lgr5 expression: results
from computational analysis
Transcriptomic and proteomic databases to compare our Lgr5 expression data (0.44-fold in comparison with the mean of our house keeping-selected genes) with those reposed in public resources were analyzed. With regard to GDS4103 microarray experiments collected in GEO section of National Center for Biotechnology Information (NCBI), we have calculated Lgr5 expression value of 0.1-fold in comparison with the mean of our HK-selected genes, both in normal and in cancer samples. Instead GDS4102 dataset points out 0.0025fold for normal and 0.0083-fold for cancer, GDS4336 gives 0.44-fold for normal and 0.51fold for cancer, dataset GDS596 reports 0.02fold in normal samples but it does not store tumor samples. In addition, according to microarray data of RefExA (Reference database for gene Expression Analysis of the University of Tokyo [21]) Lgr5 is not expressed in normal pancreas, and according to SAGE data of CGAP (Cancer Genome Anatomy Project) [22] Lgr5 is nearly absent both in normal (5 tags) and tumor (6 tags) pancreatic tissues. According to Human Protein Atlas [23,24] , in normal pancreas Lgr5 protein is expressed on average in exocrine glandular cells and highly in Islets of Langerhans. Moreover, it is expressed at an intermediate level in 60%, low in 15% and absent in the remaining 15% of pancreatic cancer samples collected in the same web repository. A mass spectrometry experiment (identifier HuPA_00784) provides the insight that protein is present in normal pancreas in Human Proteinpedia [25,26] , while this is not reported in Human Protein Reference Database [27,28] and GenAtlas [29,30] . By analyzing literature data [31,32] , it is possible to define and to draw pathways where Lgr5 may be involved. Figure 1 shows that Wnt signaling promotes Lgr5 protein expression by increasing Lgr5 gene transcription. In turn Wnt, by increasing β-catenin, could increase c-Myc transcription, resulting in a decrease of miR-23 transcription and an increase of AP4 transcription leading to an increase of Lgr5 protein expression. Furthermore, c-Myc may decrease miR-34 transcription with an increase
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Lgr5 expression, cancer stem cells & pancreatic cancer of ZNF281 and then of Lgr5. Finally, ZNF281 acts also as negative feedback since it decreases β-catenin transcription. Discussion In the last decade, increasing attention has been focused on CSCs as potential therapeutic target in several human malignancies, including PDAC. However, the identification and the characterization of CSCs remain a challenge so far. Lgr5, a Wnt target gene, has been proposed as a CSC marker in colorectal and gastric cancers [33,34] . Fujishita et al. [35] showed that Wnt/ β-catenin signaling pathway is important for the activation of the mTOR pathway during intestinal polyp formation. Moreover, Ko et al. [36] proposed Lgr5 as a potential stem cell marker in pancreatic neuroendocrine tumors. Wnt signaling pathway plays a crucial role in the initiation and progression of PDAC [37,38] . Based on this evidence, Lgr5 expression has also been investigated in pancreatic cancer. Simon et al. [39] showed higher levels of Lgr5 expression, mainly cytoplasmic, in 17 PDAC samples compared with normal pancreatic tissue. Similarly, Mizuno et al. [40] showed that Lgr5 is expressed on the cytoplasm of pancreatic cancer cells and on the basolateral membrane of endocrine pancreatic cells in patients affected by PDAC. In our study, the expression of Lgr5 in tumor samples from patients with PDAC was investigated. As previously reported, we showed that PDAC cells express Lgr5. The present results were compared with previous data on normal pancreatic tissue and we found GDS4102 dataset showing that Lgr5 is overexpressed in tumor samples. The correlation between Lgr5 expression and other pancreatic CSC markers, such as CD24, CD44, OCT3/4 and PROM1 was also evaluated. Differently from other tumors,
Research Article
a significant correlation between LGR5 and the other CSC markers was not observed. Furthermore, several studies revealed a prognostic role of Lgr5 expression in a series of solid tumors, including colorectal [10,41–42] and gastric carcinoma [43] , glioblastoma [11] , esophageal adenocarcinoma [44] and lung cancer [45] . In addition, Lgr5 expression levels have been shown to be correlated with the clinical characteristics of cancer patients, such as tumor node metastasis stage, tumor grade, lymph node metastasis and vascular invasion [11,39,44,46] . In our study, Lgr5 expression was not significantly associated with the pathological characteristics as well as with the clinical outcome of PDAC patients. Based on these results, we investigated for the biological mechanisms underlying the role of Lgr5 in PDAC. Lgr5 gene yields three alternative splicing forms (wild-type LGR5/GPR49, GPR49Δ5 and VSP_037746) characterized by altered binding affinity to their ligands [47] . Rot el al. [48] described a splice variant of Lgr5 in the context of soft tissue sarcoma and reported that low expression levels of this variant transcript (which lacks exon 5) were associated with worse overall survival. Based on this observation, further studies are needed to assess the role of different Lgr5 splicing variants in PDAC. Furthermore, the contribution of Lgr5 to cancer maintenance and progression is likely to be determined by both genetic and epigenetic state of the affected cell as well as the surrounding microenvironment. Lgr5 has been suggested to be a target gene of miR-23b [41] , a miRNA commonly expressed in pancreatic cell lines [49] . Similarly, Lgr5 seems to be also the target gene of miR-142-3p, which has been shown to inhibit the Lgr5 protein expression in colon cancer [50] . Moreover, other miRNAs could regulate Lgr5, such as miR-216a, expressed in normal pancreas [51] but downregulated in PDAC [52] or miR-342-3p overexpressed in pancreatic
Table 3. Results of univariate and multivariate regression of Lgr5 expression level and of the pathological characteristics of pancreatic ductal adenocarcinoma. Variable Gender (male vs female) Age (70 years) pT (pT1–pT2 vs pT3) pN (pN0 vs pN1) Grading (grade 1–2 vs grade 3) LGR5 expression
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Univariate
Multivariate
Hazard ratio (95% CI) p-value
Hazard ratio (95% CI) p-value
1.24 (0.73–2.12) 1.92 (1.11–3.31) 2.02 (0.72–5.62) 1.25 (0.68–2.28) 1.17 (0.74–1.85) 1.33 (0.85–2.09)
1.73 (0.97–3.09) 1.56 (0.54–4.53) 1.31 (0.85–2.03)
0.424 0.019 0.180 0.475 0.512 0.21
0.065 0.416 0.225
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Research Article Andrikou, Santoni, Piva et al.
SLUG
c-Myc
AP4
miR-23
AP4
miR-23
ZEB1
ZEB2
TWIST
c-Myc
p53
E-cadherin
miR-34 ZNF281
LGR5
SNAIL miR-34
p21 LGR5
NOTCH1
SNAIL
ZNF281
SOX4
NOTCH1 β-catenin
β-catenin
Axin2
Degradation and phosphorylation
Gastrin ODCD
Axin2 CK1α GSK3 APC
Nanog
EMT
Wnt CD44
C-Myc
TCF-1
C-jun
MMP7
CD133
Cyclin D1
TCF4
Figure 1. Pathway in pancreatic cancer cells that shows the complex regulation of Lgr5 by Wnt signaling pathway. Wnt signaling promotes Lgr5 protein expression: by increasing Lgr5 gene transcription; by increasing c-Myc transcription, resulting in a decrease of miR-23 transcription and an increase of AP4 transcription; by decreasing miR-34 transcription with an increase of ZNF281 and then of Lgr5. The rectangles and double helix stylization indicate a gene and its promoter, while smoothed rectangles indicate a protein or a transcription factor. Alone rectangles indicate mature miRNA.
tumors [53,54] . However, the mechanisms by which miRNAs induce translational repression are still not clear and deserve ulterior investigations in PDAC. The main limitations of this study include the lack of data on Lgr5 protein levels, which could not directly reflect the RNA levels due to the post-transcriptional regulation, as well as its retrospective design, which is susceptible to bias in data selection and analysis. Moreover, other markers of stemness, such as CXCR4 or Nanog, have not been evaluated in this study.
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Despite these limitations, our study shows that Lgr5 does not seem to be correlated with the pathological characteristics and outcome of patients with PDAC, as well as with the expression of other evaluated CSCs markers. Lgr5 seems to be overexpressed in PDAC, but its role remains still controversial. Conclusion Our analyses suggest that Lgr5 RNA expression in human pancreatic tumor samples does not have a prognostic role in this disease. Moreover,
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Lgr5 expression, cancer stem cells & pancreatic cancer considering the lack of association between RNA expression levels of Lgr5 and of tested pancreatic CSC markers, Lgr5 does not seem to have a role as potential CSCs marker in PDAC. Future perspective Despite the development of new therapeutic approaches, pancreatic cancer remains one of the deadliest cancer-related diseases in the world [55] . Several groups demonstrated that CSCs are responsible for the resistance of this disease to conventional treatment [56,57] . Therefore, this group of undifferentiated cells seems to be a very intriguing target for therapy and actually findings suggest that specific elimination of these cells is possible and therapeutically relevant. Indeed, recently it has been demonstrated that a multimodal treatment including two relevant CSC pathway inhibitors and addition chemotherapy represents a very promising approach with a marked survival benefit in preclinical model [58] . Whereas further studies are needed to confirm these results, a depletion of CSCs may well become a powerful target in clinical cancer therapy. Furthermore, it is important to find markers that exclusively identify pancreatic CSCs but
Research Article
Lgr5 expression does not seems to be relevant in this field. Thus, further investigation is needed to evaluate the clinical significance of these findings and an accurate study to define specific pancreatic CSC markers that may lead to the development of novel treatment regimens in this disease. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
EXECUTIVE SUMMARY ●●
Wnt signaling pathway plays a crucial role in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC).
●●
Lgr5, a Wnt target gene, has been proposed as a potential stem cell marker in various human malignancies, but its role in PDAC remains still unclear.
●●
In 69 PDAC patients underwent surgical resection, Lgr5 seems to be overexpressed in tumor samples in comparison with normal tissue.
●●
Lgr5 RNA expression was not associated with the expression of stem cell markers, such as CD24 (rho = 0.133), CD44 (rho = -0.19), OCT3/4 (rho = 0.159) and PROM1 (rho = 0.304).
●●
Lgr5 RNA expression levels were not associated with any of the pathological characteristics of PDAC.
●●
At univariate analysis Lgr5 RNA expression was not an independent predictor of survival (p = 0.21).
●●
Further studies are needed to assess the role of different Lgr5 splicing variants in PDAC.
●●
Thus, currently Lgr5 does not seem to represent a potential cancer stem cell marker in PDAC. determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1(3), 313–323 (2007).
4
Takebe N, Ivy SP. Controversies in cancer stem cells: targeting embryonic signaling pathways. Clin. Cancer Res. 16(12), 3106–3112 (2010).
•
Shows that a subpopulation of migrating CD133 + CXCR4 + cancer stem cells is essential for tumor metastasis in pancreatic ductal adenocarcinoma.
5
Penchev VR, Rasheed ZA, Maitra A, Matsui W. Heterogeneity and targeting of pancreatic cancer stem cells. Clin. Cancer Res. 18(16), 4277–4284 (2012).
3
Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell 124(6), 1111–1115 (2006).
•• This review evaluated the potential relationships between different pancreatic
References Papers of special note have been highlighted as: • of interest; •• of considerable interest 1
Siegel R, Naishadham D, Jemal A. Cancer statistics. CA Cancer J. Clin. 63(1), 11–30 (2013).
2
Hermann PC, Huber SL, Herrler T et al. Distinct populations of cancer stem cells
future science group
www.futuremedicine.com
1043
Research Article Andrikou, Santoni, Piva et al. cancer stem cell populations and strategies to identify novel targeting approaches. 6
7
8
9
Hao HX, Xie Y, Zhang Y et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485(7397), 195–200 (2012). Koo BK, Spit M, Jordens I et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488(7413), 665–669 (2012). Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc. Natl Acad. Sci. USA 108(28), 11452–11457 (2011). Clevers H, Nusse R. Wnt/β-catenin signaling and disease. Cell 149(6), 1192–1205 (2012).
10 Wu XS, Xi HQ, Chen L. Lgr5 is a potential
marker of colorectal carcinoma stem cells that correlates with patient survival. World J. Surg. Oncol. 10, 244 (2012).
specifically overexpressed in tumor epithelia. Hepatogastroenterology 55(88), 2016–2027 (2008). 19 Su AI, Wiltshire T, Batalov S et al. A gene
atlas of the mouse and human proteinencoding transcriptomes. Proc. Natl Acad. Sci. USA 101(16), 6062–6067 (2004). 20 Zhang G, Schetter A, He P et al. DPEP1
inhibits tumor cell invasiveness, enhances chemosensitivity and predicts clinical outcome in pancreatic ductal adenocarcinoma. PLoS ONE 7(2), e31507 (2012). 21 Laboratory for System Biology and Medicine.
www.lsbm.org 22 Strausberg RL1, Buetow KH, Emmert-Buck
MR, Klausner RD. The cancer genome anatomy project: building an annotated gene index. Trends Genet. 16(3), 103–106 (2000). 23 Uhlen M, Oksvold P, Fagerberg L et al.
Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28, 1248–1250 (2010).
•• In patients with colorectal cancer, Lgr5 correlates with poor prognosis and may also be considered as a potential marker for colorectal cancer stem cells.
24 Protein Atlas.
11 Nakata S, Campos B, Bageritz J et al. LGR5 is
25 Kandasamy K, Keerthikumar S, Goel R et al.
a marker of poor prognosis in glioblastoma and is required for survival of brain cancer stem-like cells. Brain Pathol. 23(1), 60–72 (2013). •
Shows that LGR5 plays an important role in maintenance and/or survival of brain CSCs in this disease.
12 SABiosciences.
www.SABiosciences.com 13 Fagerland MW, Hosmer DW, Bofin AM.
Multinomial goodness-of-fit tests for logistic regression models. Stat. Med. 27(21), 4238–4253 (2008). 14 Mickey RM, Greenland S. The impact of
confounder selection criteria on effect estimation. Am. J. Epidemiol. 129(1), 125–137 (1989). 15 Edgar R, Domrachev M, Lash AE. Gene
Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30(1), 207–210 (2002).
Human Proteinpedia: a unified discovery resource for proteomics research. Nucleic Acids Res. 37, D773–D781 (2009). 26 Human Proteinpedia.
www.humanproteinpedia.org 27 Peri S, Navarro JD, Amanchy R et al.
Development of human protein reference database as an initial platform for approaching systems biology in humans. Genome Res. 13(10), 2363–2371 (2003). 28 Human Protein Reference Database.
www.hprd.org 29 Frézal J. Genatlas database, genes and
development defects. C. R. Acad. Sci. III 321(10), 805–817 (1998). 30 Gene Atlas.
www.genatlas.org 31 Jackstadt R, Roh S, Neumann J et al. AP4 is a
mediator of epithelial-mesenchymal transition and metastasis in colorectal cancer. J. Exp. Med. 210(7), 1331–1350 (2013). 32 Hahn S, Jackstadt R, Siemens H, Hunten S,
16 NCBI.
www.ncbi.nlm.nih.gov/geo/ 17 Pei H, Li L, Fridley BL, Jenkins GD et al.
FKBP51 affects cancer cell response to chemotherapy by negatively regulating Akt. Cancer Cell 16(3), 259–266 (2009). 18 Badea L, Herlea V, Dima SO, Dumitrascu T,
Popescu I. Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes
1044
www.proteinatlas.org
Hermeking H. SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial-mesenchymal transition. EMBO J. 32(23), 3079–3095 (2013). 33 Kember K, Prasetyanti PR, De Lau W,
Rodermond H, Clevers H, Medema JP. Monoclonal antibodies against Lgr5 identify human colorectal cancer stem cells. Stem Cells 30(11), 2378–2386 (2012).
Future Oncol. (2015) 11(7)
34 Zheng ZX, Sun Y, Bu ZD et al. Intestinal
stem cell marker LGR5 expression during gastric carcinogenesis. World J. Gastroenterol. 19(46), 8714–8721 (2013). 35 Fujishita T, Aoki K, Lane HA, Aoki M,
Taketo MM. Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in ApcΔ716 mice. Proc. Natl Acad. Sci. USA 105(36), 13544–13549 (2008). 36 Ko SB, Azuma S, Yokoyama Y et al.
Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas. Am. J Physiol. Gastrointest. Liver Physiol. 304(12), G1103–G1116 (2013). 37 Pasca di Magliano M, Biankin AV, Heiser
PW et al. Common activation of canonical Wnt signaling in pancreatic adenocarcinoma. PLoS ONE 2(11), e1155 (2007). 38 Morris JP, Cano DA, Sekine S, Wang SC,
Hebrok M. Beta-catenin blocks Krasdependent reprogramming of acini into pancreatic cancer precursor lesions in mice. J. Clin. Invest. 120(2), 508–520 (2010). 39 Simon E, Petke D, Boger C et al. The spatial
distribution of LGR5+ cells correlates with gastric cancer progression. PLoS ONE 7(4), e35486 (2012). 40 Mizuno N, Yatabe Y, Hara K et al.
Cytoplasmic expression of LGR5 in pancreatic adenocarcinoma. Front. Physiol. 4, 269 (2013). •• Demonstrated that LGR5 is expressed in the cytoplasm of pancreatic cancer cells and the basolateral membrane of endocrine cells of the pancreas in patients with PDA. 41 Saigusa S, Inoue Y, Tanaka K et al. Significant
correlation between LKB1 and LGR5 gene expression and the association with poor recurrence-free survival in rectal cancer after preoperative chemoradiotherapy. J. Cancer Res. Clin. Oncol. 139(1), 131–138 (2012). 42 Takahashi H, Ishii H, Nishida N et al.
Significance of Lgr5 (+ve) cancer stem cells in the colon and rectum. Ann. Surg. Oncol. 18(4), 1166–1174 (2011). 43 Zheng ZX, Sun Y, Bu ZD et al. Intestinal
stem cell marker LGR5 expression during gastric carcinogenesis. World J. Gastroenterol. 19(46), 8714–8721 (2013). 44 Becker L, Huang Q, Mashimo H. Lgr5, an
intestinal stem cell marker, is abnormally expressed in Barrett’s esophagus and esophageal adenocarcinoma. Dis. Esophagus. 23(2), 168–174 (2010). 45 Ryuge S, Sato Y, Jiang SX et al. The
clinicopathological significance of Lgr5 expression in lung adenocarcinoma. Lung Cancer 82(1), 143–148 (2013).
future science group
Lgr5 expression, cancer stem cells & pancreatic cancer 46 Xi HQ, Cai AZ, Wu XS et al. Leucine-rich
repeat-containing G-protein-coupled receptor 5 is associated with invasion, metastasis, and could be a potential therapeutic target in human gastric cancer. Br. J. Cancer 110(8), 2011–2020 (2014). •
In this subset of patients, Lgr5 seems to be correlated with invasion and metastasis.
47 Chen PH, Chen X, Lin Z, Fang D, He X. The
structural basis of R-spondin recognition by LGR5 and RNF43. Genes Dev. 27(12), 1345–1350 (2013). 48 Rot S, Taubert H, Bache M et al. A novel
splice variant of the stem cell marker LGR5/ GPR49 is correlated with the risk of tumor-related death in soft-tissue sarcoma patients. BMC Cancer 11, 429 (2011). 49 Wang P, Zhang J, Zhang L et al. MicroRNA
23b regulates autophagy associated with radioresistance of pancreatic cancer cells. Gastroenterology 145(5), 1133–1143 (2013).
future science group
50 Shen WW, Zeng Z, Zhu WX, Fu GH.
MiR-142-3p functions as a tumor suppressor by targeting CD133, ABCG2, and Lgr5 in colon cancer cells. J. Mol. Med. (Berl). 91(8), 989–1000 (2013). 51 Endo K, Weng H, Kito N, Fukushima Y, Iwai
N. MiR-216a and miR-216b as markers for acute phased pancreatic injury. Biomed. Res. 34(4), 179–188 (2013). 52 Szafranska AE, Davison TS, John J et al.
MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene 26(30), 4442–4452 (2007). 53 Roldo C, Missiaglia E, Hagan JP et al.
MicroRNA expression abnormalities in pancreatic endocrine and acinar tumors are associated with distinctive pathologic features and clinical behavior. J. Clin. Oncol. 24(29), 4677–4684 (2006).
Research Article
54 Lee YS, Dutta A. MicroRNAs in cancer.
Annu. Rev. Pathol. 4, 199–227 (2009). 55 Philip PA, Mooney M, Jaffe D et al.
Consensus report of the national cancer institute clinical trials planning meeting on pancreas cancer treatment. J. Clin. Oncol. 27, 5660–5669 (2009). 56 Hong SP, Wen J, Bang S, Park S, Song SY.
CD44-positive cells are responsible for gemcitabine resistance in pancreatic cancer cells. Int. J. Cancer 125(10), 2323–2331 (2009). 57 Jimeno A, Feldmann G, Suarez-Gauthier A
et al. A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development. Mol. Cancer Ther. 9, 2582–2592 (2009). 58 Mueller MT, Hermann PC, Witthauer J et al.
Combined targeted treatment to eliminate tumorigenic cancer stem cells in human pancreatic cancer. Gastroenterology 137, 1102–1113 (2009).
www.futuremedicine.com
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