Notch1 pathway in adrenocortical carcinomas: correlations with clinical outcome
1Cristina L. Ronchi, 2Silviu Sbiera, 1Barbara Altieri, 1Sonja Steinhauer, 3-4Vanessa Wild, “Michaela Bekteshi, 4Matthias Kroiss, Martin Fassnacht, 1Bruno Allolio
1Department of Internal Medicine I, Endocrine and Diabetes Unit, University Hospital, University of Wuerzburg, Germany
1 2 3 4 5 6 2Central Laboratory, University Hospital of Wuerzburg, Germany 7 3Institute of Pathology, University of Wuerzburg, Germany 8 4Comprehensive Cancer Center Mainfranken, Wuerzburg, Germany
9 10 11 12 13 14 Short title: Notch1 signaling in adrenocortical tumors 15 16 Word count: 4033 17 18 19 20 21 Cristina L. Ronchi, MD, PhD 22 Unit of Endocrinology, Department of Internal Medicine I 23 University Hospital, University of Wuerzburg 24 Oberrduerrbacher-Strasse 6
Key words: adrenocortical tumors, Notch1 pathway, Jagged1, Wnt/B-catenin
25 97080 Wuerzburg (Germany)
26 Tel number: +49-0931-20139720 27 Fax: +49-0931-201639720 28 E-mail: Ronchi_C@ukw.de
1 Abstract
2 3 4 5 6 7 Previous SNP array analyses revealed genomic alterations of the Notch pathway as the most frequent abnormality in adrenocortical tumors (ACT). Aim of the study was to evaluate the expression of components of Notch signaling in ACT and to correlate them with clinical outcome. The mRNA expression of JAG1, NOTCH1, and selected target genes of Notch1 (HES1, HES5, HEY2) was evaluated in 80 fresh-frozen samples (28 normal adrenal glands=NAG, 24 adenomas=ACA, 28 carcinomas=ACC) by qRT-PCR. Immunohistochemistry was performed in 221 tissues on paraffin slides (16 NAG, 27 ACA, 178 ACC) for 8 Jagged1, activated NOTCH1 (aNOTCH1) and HEY2. An independent ACC validation cohort (n=77) was then also investigated. HEY2 mRNA expression was higher in ACC than in ACA (P<0.05). Protein expression of all factors was high (H-score 2-3) in a larger proportion of ACCs than ACAs and NAG (Jagged1 in 27%, 15% and 10%; aNOTCH1 in 13%, 8% and none; HEY2 in 66%, 61% and 33%, respectively, all P<0.001). High Jagged1 expression was associated with earlier tumor stages and lower number of metastases in ACC (both P=0.08), and impacted favorably on overall and progression free survival (131 vs 30 months, HR=0.45, and 37 vs 9 months, HR=0.51, both P<0.005). This impact on overall survival was confirmed in the validation cohort. No such association was observed for aNOTCH1 or HEY2. In conclusion, different components of the Notch1 signaling pathway are overexpressed in ACC, suggesting a role in malignant transformation. However, Jagged1 is overexpressed in a subgroup of ACCs with a better clinical outcome.
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Introduction
The pathogenesis of both benign adrenocortical adenomas (ACA) and carcinomas (ACC) remains still incompletely understood (Else, et al. 2014; Fassnacht, et al. 2013), despite major recent advances (Assie, et al. 2014). In a previous study employing SNP array profiling in adrenocortical tumors, we identified the Notch1 signaling pathway as the most frequently altered pathway in both ACA and ACC, followed by alterations in Wnt/ß-catenin signaling (Ronchi, et al. 2013). This observation suggests a major role of Notch signaling in adrenocortical tumorigenesis.
The Notch signaling pathway regulates cell-fate decisions throughout embryonic development and 9 adult life, controlling neurogenesis, angiogenesis, apoptosis, cell cycle, proliferation and differentiation (Capaccione and Pine 2013). Notch is a transmembrane receptor, with an extracellular domain possessing epidermal growth factor (EGF) repeats and an intracellular domain containing a nuclear localization sequence (NICD), a RBP-Jkappa-associated module (RAM) domain, a C-terminal PEST region, and seven ankyrin repeats. Up to now, four receptors (Notch1, Notch2, Notch3, Notch4) and six ligands (Jagged1, 14 Jagged2, Delta-like1, Delta-like3, Delta-like4, Delta-like1 homolog) have been identified. After binding of a ligand to the respective receptor, y-secretase complex mediates cleavage of the transmembrane domain of the Notch receptor to release the NICD that translocates into the nucleus and activates transcription of several target genes, including the hairy enhancer of split (HES) family, the Hes-related (HEY) family, and many others involved in apoptosis (NFKB1-2, CDKNIA, BIRC5/surviving, bcl-2), cell cycle or proliferation (cyclinD1, Deltex-1, p21/Waf1, CDKN1A-B, IGF1-R), transcription (c-myc, GATA3) or with unknown function (Notch3, PTCRA) (Supplementary Figure 1) (Chillakuri, et al. 2012; Ranganathan, et al. 2011).
Dysregulation of the Notch signaling pathway has been implicated in several human cancers. For instance, the translocation t(7;9)(q34;q34.3) or mutations in the NOTCH gene (including PEST truncating mutations), leading to Notch-ligand independent activation or to impaired degradation of activated Notch, play a role in T-cell acute lymphoblastic leukemia (Ferrando 2010; Jundt, et al. 2008; Paganin and Ferrando 2011; Weng, et al. 2004). In solid tumors, both oncogenic actions or a tumor suppressor role of the Notch signaling pathway and its components have been reported, depending on cell type and context (Supplementary Table 1) (Balint, et al. 2005; Carvalho, et al. 2014; Du, et al. 2014; Lobry, et al. 2011; Mazur, et al. 2012; Radtke and Raj 2003; Rizzo, et al. 2013; Wang, et al. 2006; Westhoff, et al. 2009). In
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most cases, activated Notch signaling is associated with more aggressive behavior and poor prognosis (Capaccione and Pine 2013). However, in some malignancies Notch1 activation may also induce cell growth arrest (Wang, et al. 2007).
Recent data suggest that Notch works as a hub enabling cross-talk among different oncogenic 5 pathways, such as the Wnt/B-catenin signaling, the Sonic-Hedgehog (Shh), and the AKT/PI3K pathways 6 7 8 9 10 11 (Supplementary Figure 1). In particular, the link between the Notch and Wnt/B-catenin signaling has been investigated in human development (Balint et al. 2005; Crosnier, et al. 2006; Gopalakrishnan, et al. 2014; Peignon, et al. 2011; Ravindran and Devaraj 2012; Yamamizu, et al. 2010) and either additive or opposing effects have been reported, depending on the respective tissue and interfering factors (Kim, et al. 2012; Kwon, et al. 2011). Interestingly, Notch pathway activation has also been related, alone or together with Wnt/B-catenin activation and p53 deletion, to the process of epithelial-to-mesenchymal transition involved in 12 initiation of metastasis (Chanrion, et al. 2014; Espinoza and Miele 2013; Wang, et al. 2009a), and to resistance to treatment (Ma, et al. 2013; Wang, et al. 2009b; Yao and Qian 2010; Yoon, et al. 2014).
Due to its oncogenic role in many cancers, inhibitors of the Notch pathway have been developed which act either at the level of gamma-secretase or bind to Notch ligands or receptors, thereby inhibiting Notch activation and suppressing tumor cell growth (Supplementary Figure 1) (Capaccione and Pine 2013; 17 Espinoza and Miele 2013; Gordon and Aster 2014; Groth and Fortini 2012; Previs, et al. 2014). Several clinical trials with these compounds alone or in combination are ongoing or have been recently completed (clinicaltrials.gov) (Lee, et al. 2015; LoConte, et al. 2015; Messersmith, et al. 2015; Richter, et al. 2014). Moreover, inhibiting Notch signaling (i.e. by pre-treatment) sensitizes tumors to platinum-compounds or other cytotoxic drugs, such as gemcitabine, (McAuliffe, et al. 2012; Meng, et al. 2009; Wang, et al. 2010). Finally, as the Notch ligand Jagged1 is over-expressed in many cancers and plays an important role in tumor biology (Steg, et al. 2011), targeting Jagged1 directly may represent a promising therapeutic tool (Dai, et al. 2014; Li, et al. 2014).
Adrenal gland morphology and functions are deeply interconnected in which signaling pathways, such as the Wnt/B-catenin, Shh and Notch, are required for their integrity (Gallo-Payet and Battista 2014). In particular, constitutive activation of the Wnt/ß-catenin signaling pathway has been demonstrated to be an early step in adrenocortical tumorigenesis (El Wakil and Lalli 2011; Gaujoux, et al. 2011; Tissier, et al.
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2005), while so far only limited data are available on the involvement of Notch signaling. However, recurrent copy number gains in JAG1 (frequency >50%) or JAG2 gene (frequency >40%) were found in ACCs suggesting a role in malignant transformation (Ronchi et al. 2013). Furthermore, Simon et al. demonstrated that JAG1 gene is over-expressed in ACC (in comparison to normal adrenals and adenomas) and that Jagged1 up-regulation in Y1 mouse cells is able to enhance cell proliferation and aggressiveness through activation of Notch signaling in adjacent cells (Simon, et al. 2012).
The major aim of the present study was, therefore, to more comprehensively investigate the 8 components of the Notch pathway and their relation to Wnt/B-catenin signaling in normal adrenal glands and in a large series of adrenocortical tumors in order to characterize a potential role in adrenocortical tumorigenesis. Furthermore, we also investigated the relationship between expression of Notch-related factors and clinical outcome in patients with ACC.
Materials and methods
Tissue samples, patients and clinical annotations
Eighty fresh frozen adrenal tissues (28 normal adrenal glands=NAG, 24 ACA and 28 ACC) were used for 16 the evaluation of mRNA levels of several components of the Notch and the Wnt/beta catenin pathways.
A series of 236 tissue samples on paraffin slides was evaluated by immunohistochemistry comprising 16 NAG (including 7 adrenal hyperplasia), 27 ACA, 178 ACC, and 15 other tissues serving as controls (including pancreas, colon, prostate and ovarian cancer). Among the ACC samples, 135 were obtained from surgery of the primary tumor, 26 from local recurrences and 17 from distant metastases. In this series, 16 ACCs had been also investigated as part of the previous SNP array analysis and were used for the comparison between CN alterations and protein expression (Ronchi et al. 2013).
Clinical parameters, such as sex, age at diagnosis, date of surgery, tumor size, results of hormone analysis, and in case of ACC, tumor stage according to the European Network for the Study of Adrenal Tumors (ENSAT) classification (Fassnacht, et al. 2009), Weiss score, Ki67 proliferation index, presence and number of distant metastases, such as detailed follow-up information were collected through the German ACC and the ENSAT Registries (www.ens@t.org/registry). Malignancy and hormonal hypersecretion were defined according to established clinical, biochemical, and morphological criteria (Nieman, et al. 2008). The
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baseline patient and tumor characteristics of both series are given in Table 1. Concerning treatment,
26 ACC patients underwent follow up only, 34 received mitotane monotherapy (adjuvant or palliative), 62 underwent chemotherapy with different drug combinations, including platinum-compound based protocols, streptozotocin, gemcitabine plus capecitabine, and others. Thirteen ACC patients were lost to follow-up (unknown treatment).
An independent validation cohort of 77 ACCs (63 obtained from surgery of the primary tumor, 6 from local recurrences and 8 from distant metastases) was also investigated in a second step by immunohistochemistry to confirm key results of the first series.
The study was approved by the ethics committee of the University of Wuerzburg (No. 93/02 and 88/11) and written informed consent was obtained from all patients.
NOTCH1 mutation analysis
A total of 46 fresh frozen adrenocortical tumors (21 ACA and 25 ACC) were investigated for the presence of 14 the somatic mutation in Exon 34.1 of the NOTCH1 gene that disrupts the PEST region (c.7544_7545delCT) leading to constitutive activation. Total tumor DNA was extracted and mutation analysis was performed by direct Sanger sequencing analysis of PCR products obtained using primer sequences and PCR conditions as previously published (Fabbri, et al. 2011; Rossi, et al. 2012).
Gene expression analysis
mRNA expression in fresh frozen tissue was investigated by real-time quantitative PCR (qRT-PCR). Among the Notch-related factors, we choose the Notch ligand Jagged1, due to the previous observations in ACT, and the most well-known Notch-specific target genes of the HES/HEY family (HES1, HES5, and HEY2) (Chillakuri et al. 2012; Ranganathan et al. 2011) and for the Wnt/B-catenin/TCF/LEF1 axis we selected the well characterized target gene LEF1. In brief, RNA was isolated from fresh frozen tissue samples using the RNeasy Lipid Tissue Minikit (Qiagen) and reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). Predesigned Taqman® gene expression assays for JAG1 (Hs01070032_m1), NOTCH1 (Hs01062014_m1), CTNNB1 (Hs00355049_m1), HES1(Hs00172878_m1), HES5 (Hs01387463_g1), HEY2 (Hs00232622_m1), and LEF1 (Hs01547250_m1) were purchased from Applied Biosystems (Darmstadt,
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Germany). Endogenously expressed B-actin (Hs9999903_m1) was used for normalization. 40 ng cDNA was used for each PCR reaction and each sample was performed in duplicate. Transcript levels were determined using the TaqMan Gene Expression Master Mix (Applied Biosystems), the CFX96 real-time thermocycler (Biorad) and Bio-Rad CFX Manager 2.0 software. Cycling conditions were 95° C for three min followed by 50 cycles of 95 ℃ for 30 sec, 60º C for 30 sec, and 72º C for 30 sec. Using the ACT method (Pfaffl 2001),
1 2 3 4 5 6 the gene expression levels were normalized to those of B-actin, as previously described (Ronchi, et al. 2012).
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Immunohistochemistry
According to previous observations and to mRNA expression results, we selected for immuno- 10 histochemistry the Notch ligand Jagged 1, the activated NOTCH1 (aNOTCH1), the specific Notch target gene HEY2. A total of 263 paraffin-embedded specimens, including 76 standard full slides and 187 samples assembled into three tissue microarrays (TMAs) were investigated by immunohistochemistry. TMA samples were included in the analysis only if two or more evaluable cores per patient were available after the staining procedure. Thus, the final series included 236 tissue samples (16 NA, 27 ACA, 178 ACC, and 15 positive controls, see above). The validation cohort consisted in 77 ACCs distributed on 3 new TMAs.
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16 Immunostaining for Jagged 1, activated NOTCH1 and HEY2: TMA and full sections were deparaffinized 17 and immunohistochemical detection was performed using an indirect immunoperoxidase technique after high temperature antigen retrieval in 10 mM citric acid monohydrate buffer (pH 6.5) in a pressure cooker for 13 min. Blocking of unspecific protein-antibody interactions was performed with 20% human AB serum in PBS for 1h at RT. Primary antibody for Jagged 1 was a monoclonal anti-rabbit antibody (Lifespan Bioscience, EPR4290), used at a dilution of 1:300 at 4° C. Primary antibody for aNOTCH1 was a polyclonal anti-rabbit antibody against the cleaved NICD (abcam, ab 8925, epitope: VLLSRKRRRQHGQC), used at a dilution of 1:200 at 4° C. Primary antibody for HEY2 was a polyclonal anti-rabbit antibody (Sigma Aldrich, HPA030205), used at a dilution of 1:100 at 4° C, together with the N-Universal Negative Control Anti- Rabbit (Dako, Glostrup, Denmark). Signal amplification was achieved by En-Vision System Labeled Polymer-HRP Anti-Rabbit (Dako) for 40 min and developed for 10 min with DAB Substrate Kit (Vector
Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Nuclei were counterstained
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with Mayer’s hematoxilin for 2 min. For positive controls, sections with colon cancer, pancreas cancer and 2 prostate cancer were chosen, while cells of the tumor stroma served as internal negative control.
All slides were analyzed independently by two investigators blinded to clinical information (C.L.R. and S.St.). Both nuclear and cytoplasmic staining was evaluated, according to the expected localization (cytoplasmatic and membranous for Jagged1, nuclear for aNOTCH1 and HEY2) and staining intensity was graded as negative (0), low (1), medium (2), or strong (3). The percentage of positive tumor cells was calculated for each specimen and scored 0 if 0% were positive, 0.1 if 1-9% were positive, 0.5 if 10-49% were positive and 1 if 50% or more were positive. A semiquantitative H-score was then calculated by multiplying the staining intensity grading score with the proportion score as described previously (Ronchi, et al. 2009). Where discrepancies were observed, results were jointly assessed by both investigators and the final score was formed by consensus. Inter-observer agreement was strong with a Pearson’s correlation coefficient of 0.83 (95%CI 0.78-0.87) for Jagged1 and 0.67 (95%CI 0.58-0.75) for aNOTCH1.
Immunostaining for ß-catenin: Immunohistochemistry for ß-catenin had been previously performed on our TMAs and the results have been already published elsewhere (Gaujoux et al. 2011; Ronchi et al. 2012). In 15 brief, primary antibody was provided by BD Bioscience (San Jose, CA, 1:400) and the nuclear staining 16 (representative of ß-catenin pathway activation) was assessed as previously described (Gaujoux et al. 2011). A total of 59 cases among those assembled in the TMAs with fewer than two evaluable cores were excluded from the final series (seven ACAs, 50 ACCs and two NAs) for a final series of 144 samples.
Statistical analysis
The Fisher’s exact test or the Chi-square test was used to investigate dichotomic variables, while a two-sided t test (or non-parametric test) was used to test continuous variables. A non-parametric Kruskal-Wallis test, followed by Bonferroni post-hoc test, was used for comparison among several groups for non-normal distributed variables. Correlations and 95% confidence intervals (95%CI) between different parameters were evaluated by linear regression analysis. Overall survival (OS) was defined as the time from the date of primary surgery to specific death or last follow-up, while progression-free survival (PFS) was defined as the time from the date of primary tumor resection to the first radiological evidence of any kind of disease progression or relapse or death. Time to progression (TTP) during therapy was defined as the time from the
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1 2 3 4 5 6 date of first administration to the first radiological evidence of any kind of disease progression or relapse or death. All survival curves were obtained by Kaplan-Meier estimates and the differences between survival curves were assessed by the log-rank (Mantel-Cox) test. For the calculation of Hazard Ratio (HR) two ACC- groups with low or high protein expression were considered (Jagged1: high expression H-score ≥1; HEY2 H- score ≥2). A multivariate regression analysis was performed by Cox proportional hazard regression model to identify those factors that might independently influence survival. Statistical analyses were made using 7 GraphPad Prism (version 5.0, La Jolla, CA, USA) and SPSS Software (PASW Version 21.0, SPSS Inc., Chicago, IL, USA). P values <0.05 were considered as statistically significant.
Results
NOTCH1 mutation analysis
None of the 46 investigated adrenocortical tumors (21 ACA and 25 ACC) had the known activating somatic mutation of the PEST region of the NOTCH1 gene (c.7544_7545delCT).
mRNA expression of JAG1 and other Notch-related factors
Considering all the 80 samples together (NAG, ACA, and ACCs), the mRNA expression of HES1 and HEY2 was positively correlated with NOTCH1 (R=0.46, P<0.005, and R=0.22, P=0.077, respectively), and also JAG1 with both HES1 and HEY2 (R=0.28, P<0.05,and R=0.24, P=0.05, respectively), and LEF1 mRNA with CTNNB1 (R=0.72, P<0.005) indicating that up-regulation of upstream signaling results in enhanced target gene expression.
HEY2 mRNA expression was significantly higher in ACC than in ACA (0.0084±0.0094 vs 0.0042±0.0060, P<0.05), while JAG1 and HES1 showed only a trend to higher levels in ACC (both P=0.13, Figure 1). The other evaluated factors (NOTCH1, HES5, CTNNB1, LEF1) were similar in the three groups (NAG, ACA, and ACC).
Concerning the relationship with clinical parameters, we observed a positive correlation between tumor size and mRNA levels of CTNNB1 (R=0.41, P<0.005), HES1 (R=0.37, P<0.01), and HEY2 (R=0.37, P<0.05). However, no other significant correlations were observed between mRNA expression of the
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investigated markers and clinical or histopathological parameters (ENSAT tumor stage, Weiss score, Ki67, number of distant metastasis and hormone secretion status).
Protein expression of Jagged 1, aNOTCH1, HEY2 and beta-catenin
Representative examples of Jagged1, aNOTCH1 and HEY2 staining in adrenal tissue are shown in Figure 2.
Both aNOTCH1 and Jagged1 staining were relatively inhomogeneous with the percentage of positive cells ranging from 10 to 85% and from 15 to 90%, respectively. In contrast, HEY2 staining had a homogeneous tissue distribution in the entire series (positive cells >50% in more than 90% of samples).
All three evaluated components of the Notch1 signaling pathway were stronger expressed in ACC than in the other subgroups. In particular, Jagged1 protein was highly expressed (H-score 2-3) in 10% of 11 NAG, 15% of ACAs and 27% of ACCs (P<0.0005 by Chi-square test) and nuclear aNOTCH1 protein in none of NAG, 8% of ACAs, and 13% of ACCs (P<0.005). HEY2 protein, which exhibited stronger staining intensity, was highly expressed (H-score 2-3) in 33% of NAG, 61% of ACAs, and 66% of ACCs (P<0.005) (Table 1). Figure 3 shows the comparison in terms of H-score values among the three groups (NAG, ACA and ACC) for Jagged1, aNOTCH1, and HEY2. On the other hand, nuclear ß-catenin expression was detected in a larger proportion of NAG and ACA than in ACC (P<0.0001).
Of note, Jagged1 expression significantly correlated with both aNOTCH1 and HEY2 in NAG and ACAs (P<0.001 and P<0.05 by Chi-square test, respectively), but not in ACC (Supplementary Figure 2A), suggesting a conserved activation of the canonical Notch1 pathway in benign tumors, but deregulated signaling in malignant tumors. Furthermore, nuclear ß-catenin expression positively correlated with Jagged1 only in ACAs (P<0.005 by Chi-square test) and with HEY2 in both ACAs and ACCs (both P<0.005, Supplementary Figure 2B).
In the ACC group, no significant difference was observed among tumors deriving from first surgery (n=140), from local recurrence (n=26) or from metastasis (n=14) for Jagged1, aNOTCH1, and HEY2 protein expression. Among primary ACCs, Jagged1 expression negatively correlated with tumor size (R=0.18, P=0.056) and showed a trend to higher expression in patients with early ENSAT tumor stages (1 to 3, P=0.08) and with a lower number of distant metastases (P=0.08). In contrast, HEY2 levels were higher in patients with metastatic tumors (P<0.01).
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Jagged1 expression was higher in the 9 ACCs affected by copy number gains in the previous SNP array analysis (22% of cases with H-score <1, 44% with H-score 1 and 33% with H-score 2-3) compared to the 7 ACCs with normal copy number (43% of cases with H-score <1, 57% with H-score 1, P<0.005, Supplementary Figure 3).
Relationship between protein expression of Jagged1, aNOTCH1 and HEY2 and clinical outcome
Among ACCs, patients with higher Jagged1 protein expression levels (H-score ≥1) had a significantly longer OS (median: 131 vs 30 months, P<0.005, HR=0.45, 95%CI=0.32-0.77) and PFS (median: 37 vs 9 months, P<0.005, HR=0.51, 95%CI=0.35-0.78, Table 2). The favorable impact of high Jagged1 expression on OS remained significant after adjustment for ENSAT tumor stage (P<0.01) and nuclear ß-catenin expression 11 (P<0.01). Interestingly, ACCs with negative nuclear ß-catenin expression (n=62) had an impressively longer OS in case of concomitant high Jagged1 (n=37, median: 126 months) than in case of concomitant low Jagged1 (n=25, median: 21 months, P<0.01, HR=0.34, 95%CI=0.09-0.64, Figure 4A).
Furthermore, the positive influence of high Jagged1 expression on clinical outcome was particularly evident in the subgroup of patients who did not receive any pharmacological treatment (n=26, P<0.05, HR=0.12, 95%CI=0.05-0.61), but not in the subgroup of patients treated with mitotane monotherapy (n=34, P=0.15, HR=0.42, 95%CI=0.19-1.27), or cytotoxic drugs (n=62, P=0.64, HR=0.87, 95%CI=0.45-1.6).
In contrast, high HEY2 protein expression (H-score 2-3) was associated with a negative prognostic role in terms of OS (median: 35 vs 86 months, P=0.10, HR=1.47, 95%CI=0.94-2.26) and PFS (median: 9 vs 31 months, P=0.13, HR=1.34, 95%CI=0.91-2.02, Table 2). The combined analysis with nuclear ß-catenin expression showed a longer OS in the subgroup of patients with negative HEY2 and negative nuclear ß- catenin (n=21, P=0.058, Figure 4B).
Nuclear aNOTCH1 protein expression had no significant impact on OS or PFS (data not shown).
A multivariate regression analysis (by Cox regression test), including the expression of Jagged1, aNOTCH1, HEY2, ß-catenin, ENSAT tumor stage and proliferation index ki67, revealed an independent impact on OS only for Jagged1 (P=0.07, HR=0.46), tumor stage (P<0.05, HR=2.79) and ki67 (P<0.005, HR=3.81).
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1 Validation ACC cohort
We also evaluated Jagged1 and HEY2 protein expression in the independent cohort of 77 ACCs in order to validate results of the first series. Here, HEY2 resulted to be similarly expressed in the first and in the new series of ACCs, while Jagged1 was even higher expressed in the validation series (P<0.05 vs first ACC series, P<0.0005 vs ACA, Supplementary Figure 4).
Moreover, despite the lower number of patients, the favourable impact of Jagged1 protein levels on overall survival remained significant in the validation cohort of ACC patients (median: 108 vs 50 months, P<0.05, HR=0.47, 95%CI=0.24-0.94). Similarly to what observed in the first series, high HEY2 expression levels were associated with trend to a worst prognosis (median overall survival: 50 vs 100 months, P=0.13, HR=1.68, 95%CI=0.86-3.37). The graphical representation of the survival analysis for Jagged1 in the first and in the validation ACC series are reported in the Figure 5.
Discussion
14 In a previous investigation employing SNP array analysis Notch signaling emerged as the most frequently altered pathway in adrenocortical neoplasias (Ronchi et al. 2013). We, therefore, have performed in this study a more detailed analysis of components of this complex pathway in adrenal tumors at different levels. We found over-expression of the Notch1 ligand Jagged1 and the downstream target of Notch1 HEY2 in ACC compared to normal adrenal glands or benign adrenocortical lesions, both at mRNA and protein level. These results were also confirmed for immunohistochemistry in an independent validation cohort of ACCs. Furthermore, activated NOTCH1 protein expression was more frequently detected in malignant ACT. However, no activating mutations in the PEST region of the NOTCH1 gene (Fabbri et al. 2011; Ferrando
2010; Jundt et al. 2008; Paganin and Ferrando 2011; Rossi et al. 2012; Weng et al. 2004) were detected in ACCs. Of note, while gene expression of Notch1 pathway components correlated significantly in normal adrenals and adenomas, this relationship was lost in ACC indicating dysregulation of Notch1 signaling in malignant tumors. Taken together these observations confirm activation of Notch1 pathway in adrenocortical neoplasias, particularly in ACC.
Over-expression of the Notch ligand Jagged1 was related to a copy number gain of the JAG1 gene in the subset of tumors for which SNP array data were available, suggesting that this mechanism may at least in
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part underlie the increased expression of Jagged1. However, while high expression of Jagged1 was more often found in ACC than in ACA or NAG, it was characteristic of a subgroup of ACCs with a more differentiated phenotype and better prognosis. In particular, high Jagged1 expression tended to be more frequent in smaller tumors, in early tumor stages, and in tumors with lower number of distant metastasis. Furthermore, it was associated with a better prognosis in terms of both improved PFS and OS. The favorable impact of Jagged1 expression on general clinical outcome was also confirmed in the smaller validation ACC 7 cohort. Therefore, high Jagged1 indicates a less detrimental prognosis in ACC.
Higher expression of Jagged1 in ACC compared to ACA and normal adrenals has already been described by Simon et al (Simon et al. 2012), but no prognostic impact was reported. Instead, the Authors showed that Jagged1 overexpression in murine Y1 cells leads to enhanced cell proliferation and aggressiveness. This discrepancy may be explained at least in part by the specific cellular context of Y1 cells and species differences.
Intriguingly, the favorable impact of high Jagged1 expression on clinical outcome was particularly evident in tumors with negative nuclear ß-catenin expression, supporting the importance of the well described interconnection of the Wnt/B-catenin pathway (Balint et al. 2005; Crosnier et al. 2006; Gopalakrishnan et al. 2014; Kim et al. 2012; Kwon et al. 2011; Peignon et al. 2011; Ravindran and Devaraj 2012; Wang et al. 2009a; Yamamizu et al. 2010).
Different from Jagged1, high HEY2 protein expression was rather a marker of a more aggressive tumor type with inferior clinical outcome, although the impact on overall survival was not significant. This 20 discrepancy with the Jagged1 findings may reflect the profound dysregulation of the Notch pathway in ACC and the role of additional influences downstream of Notch. The demonstration of Notch activation and over- expression of its target genes in patients with advanced ACC may become therapeutically relevant, considering the potential use of Notch-inhibiting drugs acting downstream of Jagged1, like gamma-secretase inhibitors, which are currently under investigation alone or in combination in clinical trials for Notch- dependent solid tumors (Lee et al. 2015; LoConte et al. 2015; Messersmith et al. 2015; Richter et al. 2014), or more innovative compounds, such as receptor blocking monoclonal antibodies (Supplementary Figure 1) (Hernandez Tejada, et al. 2014).
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While this investigation is the first attempt to analyze in more detail the Notch pathway in adrenocortical neoplasia, there remain clear limitations. The Notch pathway is highly complex with four receptors, multiple ligands beyond Jagged1 and several direct downstream targets beyond the best-described Notch-HES/HEY family (Iso, et al. 2003) that also participate in other important signaling pathways, such as 5 the Wnt/B-catenin pathway (e.g. c-myc, cyclinD1, and survivin) (Borggrefe and Oswald 2009). Moreover, it interacts at different levels with other oncogenic pathways, such as the Wnt/B-catenin signaling, the Shh, and the AKT/PI3K pathways (Supplementary Figure 1). Thus, a complete analysis would require inclusion of many more components. However, the parameters evaluated here are established key components of this pathway allowing first important insights into Notch signaling in adrenal neoplasias.
In summary, activation of Notch1 signaling is demonstrated in ACC, suggesting a potential role in malignant adrenocortical transformation. However, Jagged1 is overexpressed in a subgroup of ACCs characterized by a more differentiated phenotype and a better clinical outcome.
Declaration of interest The Authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
18 Funding
This work was supported by a grant from the Wilhelm Sander Foundation (grant # 2012.095.1 to B.A.).
Furthermore, support was received by the Deutsche Forschungsgemeinschaft DFG (grant numbers KR- 43741/1-1 to M.K. and FA 466/4-1 to M.F.) and by the IZKF Würzburg (grant # B-281 to M.F.).
Acknowledgements
The authors are grateful to Martina Zink for excellent technical support and Michaela Haaf for coordinating the German ACC Registry.
19 20 21 22 23 24 25 26 27 28
1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17
1 References
2 Assie G, Letouze E, Fassnacht M, Jouinot A, Luscap W, Barreau O, Omeiri H, Rodriguez S, 3 Perlemoine K, Rene-Corail F, et al. 2014 Integrated genomic characterization of adrenocortical carcinoma. Nat Genet 46 607-612.
4
5 Balint K, Xiao M, Pinnix CC, Soma A, Veres I, Juhasz I, Brown EJ, Capobianco AJ, Herlyn M & 6 Liu ZJ 2005 Activation of Notch1 signaling is required for beta-catenin-mediated human primary melanoma progression. J Clin Invest 115 3166-3176.
9 10
7 8 Borggrefe T & Oswald F 2009 The Notch signaling pathway: transcriptional regulation at Notch target genes. Cell Mol Life Sci 66 1631-1646.
11
Capaccione KM & Pine SR 2013 The Notch signaling pathway as a mediator of tumor survival. Carcinogenesis 34 1420-1430.
12 Carvalho FL, Simons BW, Eberhart CG & Berman DM 2014 Notch signaling in prostate cancer: a 13 moving target. Prostate 74 933-945.
14 Chanrion M, Kuperstein I, Barriere C, El Marjou F, Cohen D, Vignjevic D, Stimmer L, Paul-
15 Gilloteaux P, Bieche I, Tavares Sdos R, et al. 2014 Concomitant Notch activation and p53 deletion 16 trigger epithelial-to-mesenchymal transition and metastasis in mouse gut. Nat Commun 5 5005. 17 Chillakuri CR, Sheppard D, Lea SM & Handford PA 2012 Notch receptor-ligand binding and activation: insights from molecular studies. Semin Cell Dev Biol 23 421-428.
18 19 Crosnier C, Stamataki D & Lewis J 2006 Organizing cell renewal in the intestine: stem cells,
20 signals and combinatorial control. Nat Rev Genet 7 349-359.
21 Dai Y, Wilson G, Huang B, Peng M, Teng G, Zhang D, Zhang R, Ebert MP, Chen J, Wong BC, et 22 al. 2014 Silencing of Jagged1 inhibits cell growth and invasion in colorectal cancer. Cell Death Dis 5 e1170.
23 24 Du X, Cheng Z, Wang YH, Guo ZH, Zhang SQ, Hu JK & Zhou ZG 2014 Role of Notch signaling 25 pathway in gastric cancer: a meta-analysis of the literature. World J Gastroenterol 20 9191-9199.
26 El Wakil A & Lalli E 2011 The Wnt/beta-catenin pathway in adrenocortical development and cancer. Mol Cell Endocrinol 332 32-37.
27 28 Else T, Kim AC, Sabolch A, Raymond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano 29 TJ & Hammer GD 2014 Adrenocortical carcinoma. Endocr Rev 35 282-326.
30 31 Espinoza I & Miele L 2013 Notch inhibitors for cancer treatment. Pharmacol Ther 139 95-110.
33
Fabbri G, Rasi S, Rossi D, Trifonov V, Khiabanian H, Ma J, Grunn A, Fangazio M, Capello D, Monti S, et al. 2011 Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation. J Exp Med 208 1389-1401.
32
34 35 36 Fassnacht M, Johanssen S, Quinkler M, Bucsky P, Willenberg HS, Beuschlein F, Terzolo M, Mueller HH, Hahner S, Allolio B, et al. 2009 Limited prognostic value of the 2004 International Union Against Cancer staging classification for adrenocortical carcinoma: proposal for a Revised 37 TNM Classification. Cancer 115 243-250.
38
Fassnacht M, Kroiss M & Allolio B 2013 Update in adrenocortical carcinoma. J Clin Endocrinol Metab 98 4551-4564.
39 40 Ferrando A 2010 NOTCH mutations as prognostic markers in T-ALL. Leukemia 24 2003-2004. Gallo-Payet N & Battista MC 2014 Steroidogenesis-adrenal cell signal transduction. Compr Physiol 4 889-964.
41 42 43 Gaujoux S, Grabar S, Fassnacht M, Ragazzon B, Launay P, Libe R, Chokri I, Audebourg A, Royer 44 B, Sbiera S, et al. 2011 beta-catenin activation is associated with specific clinical and pathologic 45 characteristics and a poor outcome in adrenocortical carcinoma. Clin Cancer Res 17 328-336.
46 Gopalakrishnan N, Saravanakumar M, Madankumar P, Thiyagu M & Devaraj H 2014
47 Colocalization of beta-catenin with Notch intracellular domain in colon cancer: a possible role of 48 Notch1 signaling in activation of CyclinD1-mediated cell proliferation. Mol Cell Biochem 396 281-
49 293.
Gordon WR & Aster JC 2014 Application and evaluation of anti-Notch antibodies to modulate Notch signaling. Methods Mol Biol 1187 323-333.
6 7 8 9 10
1 2 3 Groth C & Fortini ME 2012 Therapeutic approaches to modulating Notch signaling: current challenges and future prospects. Semin Cell Dev Biol 23 465-472.
4 5 Hernandez Tejada FN, Galvez Silva JR & Zweidler-Mckay PA 2014 The challenge of targeting notch in hematologic malignancies. Front Pediatr 2 54.
Iso T, Kedes L & Hamamori Y 2003 HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 194 237-255.
Jundt F, Schwarzer R & Dorken B 2008 Notch signaling in leukemias and lymphomas. Curr Mol Med 8 51-59.
11 12 13 14 15 16 17
Kim HA, Koo BK, Cho JH, Kim YY, Seong J, Chang HJ, Oh YM, Stange DE, Park JG, Hwang D, et al. 2012 Notch1 counteracts WNT/beta-catenin signaling through chromatin modification in colorectal cancer. J Clin Invest 122 3248-3259.
Kwon C, Cheng P, King IN, Andersen P, Shenje L, Nigam V & Srivastava D 2011 Notch post- translationally regulates beta-catenin protein in stem and progenitor cells. Nat Cell Biol 13 1244- 1251.
18
Lee SM, Moon J, Redman BG, Chidiac T, Flaherty LE, Zha Y, Othus M, Ribas A, Sondak VK, Gajewski TF, et al. 2015 Phase 2 study of RO4929097, a gamma-secretase inhibitor, in metastatic melanoma: SWOG 0933. Cancer 121 432-440.
19 20 Li D, Masiero M, Banham AH & Harris AL 2014 The notch ligand JAGGED1 as a target for anti- tumor therapy. Front Oncol 4 254. 21 22 Lobry C, Oh P & Aifantis I 2011 Oncogenic and tumor suppressor functions of Notch in cancer: it’s NOTCH what you think. J Exp Med 208 1931-1935. 23 24 LoConte NK, Razak AR, Ivy P, Tevaarwerk A, Leverence R, Kolesar J, Siu L, Lubner SJ, Mulkerin DL, Schelman WR, et al. 2015 A multicenter phase 1 study of gamma -secretase inhibitor 25 26 RO4929097 in combination with capecitabine in refractory solid tumors. Invest New Drugs 33 169- 176. 27 28 Ma Y, Ren Y, Han EQ, Li H, Chen D, Jacobs JJ, Gitelis S, O’Keefe RJ, Konttinen YT, Yin G, et al. 29 2013 Inhibition of the Wnt-beta-catenin and Notch signaling pathways sensitizes osteosarcoma cells 30 to chemotherapy. Biochem Biophys Res Commun 431 274-279.
31 Mazur PK, Riener MO, Jochum W, Kristiansen G, Weber A, Schmid RM & Siveke JT 2012
32 Expression and clinicopathological significance of notch signaling and cell-fate genes in biliary 33 tract cancer. Am J Gastroenterol 107 126-135.
34 McAuliffe SM, Morgan SL, Wyant GA, Tran LT, Muto KW, Chen YS, Chin KT, Partridge JC, Poole BB, Cheng KH, et al. 2012 Targeting Notch, a key pathway for ovarian cancer stem cells, sensitizes tumors to platinum therapy. Proc Natl Acad Sci U S A 109 E2939-2948.
35 36 37 38 39 40 Messersmith WA, Shapiro GI, Cleary JM, Jimeno A, Dasari A, Huang B, Shaik MN, Cesari R,
Meng RD, Shelton CC, Li YM, Qin LX, Notterman D, Paty PB & Schwartz GK 2009 gamma- Secretase inhibitors abrogate oxaliplatin-induced activation of the Notch-1 signaling pathway in colon cancer cells resulting in enhanced chemosensitivity. Cancer Res 69 573-582.
41 Zheng X, Reynolds JM, et al. 2015 A Phase I, Dose-Finding Study in Patients with Advanced Solid Malignancies of the Oral gamma-Secretase Inhibitor PF-03084014. Clin Cancer Res 21 60-67.
42 43 Nieman LK, Biller BM, Findling JW, Newell-Price J, Savage MO, Stewart PM & Montori VM 44 45 2008 The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab 93 1526-1540.
46 Paganin M & Ferrando A 2011 Molecular pathogenesis and targeted therapies for NOTCH1- 47 induced T-cell acute lymphoblastic leukemia. Blood Rev 25 83-90.
48 Peignon G, Durand A, Cacheux W, Ayrault O, Terris B, Laurent-Puig P, Shroyer NF, Van
49 Seuningen I, Honjo T, Perret C, et al. 2011 Complex interplay between beta-catenin signalling and 50 Notch effectors in intestinal tumorigenesis. Gut 60 166-176.
1 Pfaffl MW 2001 A new mathematical model for relative quantification in real-time RT-PCR.
2 Nucleic Acids Res 29 45.
3 Previs RA, Coleman RL, Harris AL & Sood AK 2014 Molecular Pathways: Translational and Therapeutic Implications of the Notch Signaling Pathway in Cancer. Clin Cancer Res.
4
5 Radtke F & Raj K 2003 The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer 3 756-767.
6 7 Ranganathan P, Weaver KL & Capobianco AJ 2011 Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer 11 338-351.
10
8 9 Ravindran G & Devaraj H 2012 Aberrant expression of beta-catenin and its association with DeltaNp63, Notch-1, and clinicopathological factors in oral squamous cell carcinoma. Clin Oral Investig 16 1275-1288.
11
12 Richter S, Bedard PL, Chen EX, Clarke BA, Tran B, Hotte SJ, Stathis A, Hirte HW, Razak AR, Reedijk M, et al. 2014 A phase I study of the oral gamma secretase inhibitor R04929097 in combination with gemcitabine in patients with advanced solid tumors (PHL-078/CTEP 8575). Invest New Drugs 32 243-249.
17
13 14 15 16 Rizzo P, Miele L & Ferrari R 2013 The Notch pathway: a crossroad between the life and death of the endothelium. Eur Heart J 34 2504-2509.
18 Ronchi CL, Sbiera S, Kraus L, Wortmann S, Johanssen S, Adam P, Willenberg HS, Hahner S, 19 Allolio B & Fassnacht M 2009 Expression of excision repair cross complementing group 1 and prognosis in adrenocortical carcinoma patients treated with platinum-based chemotherapy. Endocr Relat Cancer 16 907-918.
20 21 22 Ronchi CL, Sbiera S, Leich E, Henzel K, Rosenwald A, Allolio B & Fassnacht M 2013 Single 23 24 nucleotide polymorphism array profiling of adrenocortical tumors — evidence for an adenoma carcinoma sequence? PLoS One 8 e73959.
25 Ronchi CL, Sbiera S, Leich E, Tissier F, Steinhauer S, Deutschbein T, Fassnacht M & Allolio B 2012 Low SGK1 expression in human adrenocortical tumors is associated with ACTH-independent glucocorticoid secretion and poor prognosis. J Clin Endocrinol Metab 97 E2251-2260.
26 27 28 29 30
Rossi D, Rasi S, Fabbri G, Spina V, Fangazio M, Forconi F, Marasca R, Laurenti L, Bruscaggin A, Cerri M, et al. 2012 Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia. Blood 119 521-529.
Simon DP, Giordano TJ & Hammer GD 2012 Upregulated JAG1 enhances cell proliferation in adrenocortical carcinoma. Clin Cancer Res 18 2452-2464.
Steg AD, Katre AA, Goodman B, Han HD, Nick AM, Stone RL, Coleman RL, Alvarez RD, Lopez- Berestein G, Sood AK, et al. 2011 Targeting the notch ligand JAGGED1 in both tumor cells and stroma in ovarian cancer. Clin Cancer Res 17 5674-5685.
Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagnere AM, Rene-Corail F, Jullian E, Gicquel C, Bertagna X, et al. 2005 Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors.
31 32 33 34 35 36 37 38 39 Cancer Res 65 7622-7627. 40 Wang L, Qin H, Chen B, Xin X, Li J & Han H 2007 Overexpressed active Notch1 induces cell growth arrest of HeLa cervical carcinoma cells. Int J Gynecol Cancer 17 1283-1292.
41 42 Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D & Sarkar FH 2010 Targeting Notch 43 signaling pathway to overcome drug resistance for cancer therapy. Biochim Biophys Acta 1806 258- 267.
44 45 Wang Z, Li Y, Banerjee S & Sarkar FH 2009a Emerging role of Notch in stem cells and cancer. Cancer Lett 279 8-12.
46 47 Wang Z, Li Y, Kong D, Banerjee S, Ahmad A, Azmi AS, Ali S, Abbruzzese JL, Gallick GE & Sarkar FH 2009b Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine- resistant pancreatic cancer cells is linked with activation of the notch signaling pathway. Cancer
48 49 50 Res 69 2400-2407.
1 Wang Z, Zhang Y, Li Y, Banerjee S, Liao J & Sarkar FH 2006 Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells. Mol Cancer Ther 5 483-493.
2
3
4 Weng AP, Ferrando AA, Lee W, Morris JPt, Silverman LB, Sanchez-Irizarry C, Blacklow SC, 5 6 Look AT & Aster JC 2004 Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306 269-271.
7 Westhoff B, Colaluca IN, D’Ario G, Donzelli M, Tosoni D, Volorio S, Pelosi G, Spaggiari L, Mazzarol G, Viale G, et al. 2009 Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci U S A 106 22293-22298.
8
9 10 Yamamizu K, Matsunaga T, Uosaki H, Fukushima H, Katayama S, Hiraoka-Kanie M, Mitani K & 11 12 Yamashita JK 2010 Convergence of Notch and beta-catenin signaling induces arterial fate in vascular progenitors. J Cell Biol 189 325-338.
13 Yao J & Qian C 2010 Inhibition of Notch3 enhances sensitivity to gemcitabine in pancreatic cancer through an inactivation of PI3K/Akt-dependent pathway. Med Oncol 27 1017-1022.
14 15 Yoon SO, Zapata MC, Singh A, Jo WS, Spencer N & Choi YS 2014 Gamma secretase inhibitors 16 enhance vincristine-induced apoptosis in T-ALL in a NOTCH-independent manner. Apoptosis 19
17 1616-1626.
| A: Cohort for mRNA analysis by quantitative RT-PCR | ||||
|---|---|---|---|---|
| Normal adrenal | Adenoma | Carcinoma | P | |
| N | 28 | 24 | 28 | |
| Age - yrs (median, range) | - | 50.5 (25-62) | 50 (24-78) | NS |
| Sex (M/F) | - | 9/15 | 14/14 | NS |
| Tumor size - cm (median, range) | - | 3.2 (0.9-8.5) | 8 (2.5-24) | <0.05 |
| Hormone-secretion (nº available) | - | 24 | 28 | NS |
| Aldosterone-producing | - | 6 | 1 | NS |
| Cortisol-producing | - | 11 | 12 | NS |
| Androgen-producing | - | 0 | 3 | NS |
| Inactive | - | 7 | 12 | NS |
| Primary tumors | - | - | 19 | - |
| Local recurrence | - | - | 4 | - |
| Metastases | - | - | 3 | - |
| ENSAT tumor stage | - | - | ||
| ENSAT 1-2/3/4 (nº) | - | 13/10/4 | - | |
| Proliferation index (ki67) - % (median, range) | - | - | 17±10.5 | - |
| Weiss score (median, range) | - | - | 6±2 | - |
| B: Cohort for protein analysis by immunohistochemistry | ||||
| N | 16 | 27 | 178 | |
| Age - yrs (median, range) | 64 (27-79) | 48 (31-76) | 47 (18-77) | NS |
| Sex (M/F) | - | 11/17 | 56/99 | NS |
| Tumor size - cm (median, range) | - | 2.8 (1-6.5) | 11 (3-30) | NS |
| Hormone-secretion (nº available) | - | 28 | 102 | NS |
| Androgen-producing | 0 | 14 | NS | |
| Aldosterone-producing | - | 6 | 5 | NS |
| Cortisol-producing | - | 15 | 28 | NS |
| Mixed | 0 | 36 | NS | |
| Inactive | - | 7 | 19 | NS |
| Primary tumors (nº) | - | - | 135 | - |
| Local recurrence (nº) | - | - | 26 | - |
| Metastases (nº) | - | - | 17 | - |
| ENSAT tumor stage (nº) | ||||
| ENSAT 1-2/3/4 (nº) | - | - | 63/41/37 | - |
| Proliferation index (ki67) - % (median, range) | - | - | 10 (1-60) | - |
| Weiss score (median, range) | - | - | 5 (2-9) | - |
| Jagged1 protein expression (%) | <0.005 | |||
| Absent (H-score 0-0.5) | 40 | 46 | 26 | |
| Low (H-score 1) | 50 | 38 | 47 | |
| High (H-score 2-3) | 10 | 15 | 27 | |
| Notch1 protein expression (%) | <0.005 | |||
| Absent (H-score 0-0.5) | 80 | 48 | 58 | |
| Low (H-score 1) | 20 | 44 | 29 | |
| High (H-score 2-3) | 0 | 8 | 13 | |
| HEY2 protein expression (%) | <0.005 | |||
| Absent (H-score 0-0.5) | 27 | 14 | 9 | |
| Low (H-score 1) | 40 | 24 | 25 | |
| High (H-score 2-3) | 33 | 61 | 66 | |
| n | Median survival (months) | HR (95% CI) | P | |
|---|---|---|---|---|
| Overall survival | ||||
| aNotch1 | 0.80 | 0.30 | ||
| Low (H-score <1) | 82 | 35 | (0.53-1.21) | |
| High | 57 | 45 | ||
| Jagged 1 | 0.45 | <0.005 | ||
| Low (H-score 0-1) | 101 | 30 | (0.32-0.77) | |
| High (H-score 2-3) | 36 | 131 | ||
| HEY2 | 1.47 | 0.10 | ||
| Low | 44 | 86 | (0.94-2.26) | |
| High | 83 | 35 | ||
| Nuclear beta-catenin | 1.71 | <0.05 | ||
| Low | 53 | 89 | (1.05-2.99) | |
| High | 42 | 38 | ||
| Free progression survival | ||||
| aNotch1 | 0.86 | 0.41 | ||
| Low (H-score <1) | 82 | 13 | (0.58-1.23) | |
| High | 57 | 10 | ||
| Jagged 1 | 0.51 | <0.005 | ||
| Low (H-score 0-1) | 101 | 9 | (0.35-0.78) | |
| High (H-score 2-3) | 36 | 37 | ||
| HEY2 | 1.37 | 0.13 | ||
| Low | 44 | 31 | (0.92-2.07) | |
| High | 83 | 9 | ||
| Nuclear beta-catenin | 1.8 | <0.05 | ||
| Low | 53 | 29 | (1.17-3.12) | |
| High | 42 | 9 | ||
HR=hazard ratio, 95%CI=95% confidence interval.
*only ACC samples with complete available clinical data (total n=137).
1 Legend to the Figures
Figure 1. Relative mRNA Expression of JAG1, HES1 and HEY2 in normal adrenal glands (NAG, n=28), adenomas (ACA, n=24) and carcinoma (ACC, n=28).
Figure 2. Examples of immunostaining of A) activated Notch1 (aNotch1), B) Jagged1 and C) HEY2 in a normal adrenal gland and an adrenocortical carcinoma (H-score =2).
Figure 3. Evaluation of immunostaining as single and mean H-score values (±standard error) in normal adrenal glands (NAG, n=16), adrenocortical adenomas (ACA, n=27) and carcinomas (ACC, n=178). A) activated Notch1 (aNotch1), B) Jagged1, and C) HEY2. Statistical analysis by one-way ANOVA.
Figure 4. Overall survival analysis by Kaplan-Meyer curves in adrenocortical carcinomas: combination 15 between A) nuclear beta catenin and Jagged1 protein expression (n=95) and B) nuclear beta catenin and HEY2 protein expression (n=90).
Figure 5. Overall survival analysis by Kaplan-Meyer curves in the first series of adrenocortical carcinomas (ACCs, A and C, n=137) and in the validation series (B and D, n=77) for Jagged1 protein expression (A and B) and HEY2 protein expression (C and D).
2 3 4 5 6 7 8 9 10 11 12 13 14
16 17 18 19 20 21 22 23 24 25 26 27 28
1 Supplementary Figures
2
3 Supplementary Figure 1. Simplified diagram of the Notch signaling canonical pathway, including sites of potential therapeutic targets (red arrows=Notch-inhibitors, green arrows=Notch-activators), known interactions with other pathways highlighted on the left, and known functions of the Notch downstream target genes highlighted on the right.
Supplementary Figure 2. Correlation between the protein expression (evaluated as H-score) of different parameters in adrenocortical adenomas and carcinomas:
4 5 6 7 8 9 10 A. Jagged1 vs activated Notch1 (aNotch1) and HEY2 11 B. Nuclear beta-catenin vs Jagged1 and HEY2 12 13 14 carcinoma samples with normal JAG1 copy number profile (n=7) and JAG1 copy number gains (n=9) at a previous SNP array analysis (Ronchi et al. 2013).
Supplementary Figure 3. Correlation between Jagged1 protein expression (as H-score) in adrenocortical
15
Supplementary Figure 4. Evaluation of immunostaining as single and mean H-score values (±standard error) in normal adrenal glands (NAG, n=16), adenomas (ACA, n=27), first series of carcinomas (ACC, n=178) and validation series of carcinomas (validation ACC, n=77). A) Jagged1, and B) HEY2. Statistical analysis by one-way ANOVA.
16
P<0.05
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HES1 mRNA expression
1.5
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HEY2 mRNA expression
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8
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Figure 1. Relative mRNA Expression of JAG1, HES1 and HEY2 in normal adrenal glands (NAG, n=28), adenomas (ACA, n=24) and carcinoma (ACC, n=28).
56×21mm (600 x 600 DPI)
Figure 2
Normal adrenal (H-score=2)
ACC (H-score=2)
A) aNotch 1
B) Jagged 1
Normal adrenal (H-score=2) 15325
ACC (H-score=2) 886/09
capsule
zona glomerulosa (mineralcorticoids)
zona fasciculata (glucocorticoids)
zona reticularis (androgens)
C) HEY2
Normal adrenal (H-score=2) 15325
ACC (H-score=2) 28330
Figure 3
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3
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ACC
A)
B)
o- b-cat neg Jag pos
-o- b-cat neg HEY neg
Percent overall survival
100
b-cat pos Jag pos
Percent overall survival
100
b-cat neg HEY pos
b-cat neg Jag neg
b-cat pos Jag neg
b-cat pos HEY neg
75
75
b-cat pos HEY pos
50
2000
o
50
0-8000
P=0.0578
25
P=0.0027
25
o
0
0
100
200
300
0
Time (months)
0
100
200
300
Time (months)
Figure 4. Overall survival analysis by Kaplan-Meyer curves in adrenocortical carcinomas: combination between A) nuclear beta catenin and Jagged1 protein expression (n=95) and B) nuclear beta catenin and HEY2 protein expression (n=90). 49×20mm (600 x600 DPI)
FIRST SERIES (n=137)
VALIDATION SERIES (n=77)
A)
B)
Percent overall survival
100
-o- Jagged 1 H-score 0-0.5
Percent overall survival
100
-o- Jagged1 H-score 0-1
-0- Jagged H-score 1
+ Jagged1 H-score 2-3
75
+ Jagged H-score 2-3
75
P=0.036
50
P=0.0037
50
25
25
0
0
0
100
200
300
0
100
200
300
400
Time (months)
Time (months)
C)
D)
Percent overall survival
100
HEY2 h-score 0-1
-o- HEY2 h-score 0-1
HEY2 h-score 2
Percent overall survival
100
- HEY2 h-score 2-3
75
HEY2 h-score 3
75
50
P=0.048
50
P=0.13
25
o
o
25
0
0
0
100
200
300
0
100
200
300
400
Time (months)
Time (months)
Figure 5. Overall survival analysis by Kaplan-Meyer curves in the first series of adrenocortical carcinomas (ACCs, A and C, n=137) and in the validation series (B and D, n=77) for Jagged1 protein expression (A and B) and HEY2 protein expression (C and D). 81×60mm (600 x 600 DPI)