Dlk1是一种新的肾上腺皮质干细胞/祖细胞标志物,可预测肾上腺皮质癌的恶性程度。

IF 20.1 1区 医学 Q1 ONCOLOGY
Katia Mariniello, James F. H. Pittaway, Barbara Altieri, Kleiton Silva Borges, Irene Hadjidemetriou, Claudio Ribeiro, Gerard Ruiz-Babot, David S. Tourigny, Jiang A. Lim, Julie Foster, Julie Cleaver, Jane Sosabowski, Nafis Rahman, Milena Doroszko, Constanze Hantel, Sandra Sigala, Andrea Abate, Mariangela Tamburello, Katja Kiseljak-Vassiliades, Margaret Wierman, Charlotte Hall, Laila Parvanta, Tarek E. Abdel-Aziz, Teng-Teng Chung, Aimee Di Marco, Fausto Palazzo, Celso E. Gomez-Sanchez, David R. Taylor, Oliver Rayner, Cristina L. Ronchi, Carles Gaston-Massuet, Silviu Sbiera, William M. Drake, Emanuel Rognoni, Matthias Kroiss, David T. Breault, Martin Fassnacht, Leonardo Guasti
{"title":"Dlk1是一种新的肾上腺皮质干细胞/祖细胞标志物,可预测肾上腺皮质癌的恶性程度。","authors":"Katia Mariniello,&nbsp;James F. H. Pittaway,&nbsp;Barbara Altieri,&nbsp;Kleiton Silva Borges,&nbsp;Irene Hadjidemetriou,&nbsp;Claudio Ribeiro,&nbsp;Gerard Ruiz-Babot,&nbsp;David S. Tourigny,&nbsp;Jiang A. Lim,&nbsp;Julie Foster,&nbsp;Julie Cleaver,&nbsp;Jane Sosabowski,&nbsp;Nafis Rahman,&nbsp;Milena Doroszko,&nbsp;Constanze Hantel,&nbsp;Sandra Sigala,&nbsp;Andrea Abate,&nbsp;Mariangela Tamburello,&nbsp;Katja Kiseljak-Vassiliades,&nbsp;Margaret Wierman,&nbsp;Charlotte Hall,&nbsp;Laila Parvanta,&nbsp;Tarek E. Abdel-Aziz,&nbsp;Teng-Teng Chung,&nbsp;Aimee Di Marco,&nbsp;Fausto Palazzo,&nbsp;Celso E. Gomez-Sanchez,&nbsp;David R. Taylor,&nbsp;Oliver Rayner,&nbsp;Cristina L. Ronchi,&nbsp;Carles Gaston-Massuet,&nbsp;Silviu Sbiera,&nbsp;William M. Drake,&nbsp;Emanuel Rognoni,&nbsp;Matthias Kroiss,&nbsp;David T. Breault,&nbsp;Martin Fassnacht,&nbsp;Leonardo Guasti","doi":"10.1002/cac2.70012","DOIUrl":null,"url":null,"abstract":"<p>Adrenocortical carcinoma (ACC) is a rare malignancy with no widely available biomarkers and commonly presents at later stages with a bleak prognosis [<span>1</span>]. Dysregulation of signaling pathways involved in the organogenesis and homeostasis of the adrenal cortex is implicated in its pathogenesis [<span>2</span>]. The paternally expressed, cleavable protein delta-like non-canonical Notch ligand 1 (DLK1) is expressed in rat adrenocortical progenitor cells [<span>3</span>] and in clusters of relatively undifferentiated cells in the human adrenal gland [<span>4</span>]. Its expression is rare in most adult human tissues but has been reported across various cancers, often associated with worse survival [<span>5</span>]. Here we define the role of DLK1 in adrenocortical development, self-renewal, and the development and progression of ACC.</p><p>Dlk1<sup>+</sup> cells were present in both the capsule and cortex during embryonic development but became restricted to the capsule postnatally in both male and female mice (Supplementary Figure S1), with minimal overlap in expression with Axin-2 (Wnt-active) cells, their early descendants, and platelet-derived growth factor receptor alpha (PDGFRα), a marker of mesenchymal stem/fibroblastic cells (Supplementary Figure S2). Dlk1 cells were rarely positive for Ki-67, whereas <i>Gli1</i> expression in the capsule, unlike Dlk1, remained high during development and throughout postnatal life (Supplementary Figure S3). Genetic lineage tracing using inducible <i>Dlk1<sup>CreERT2/+</sup></i>; <i>Rosa<sup>tdTomato/+</sup></i> mice showed that Dlk1<sup>+</sup> cells functioned as adrenocortical stem cells during development (Figure 1A-F), but were largely dormant postnatally and inactive during postnatal adrenocortical remodeling (Supplementary Figure S4).</p><p>Capsular-like cells are pathognomonic of subcapsular hyperplasia (SH), a histological hallmark in mouse adrenals that occurs spontaneously in aged females and in certain strains/transgenic models after gonadectomy (GDX) [<span>6</span>]. SH foci are thought to represent a morphological continuum progressing toward adrenocortical tumors. Dlk1 was not expressed in SH or in subsequent tumors in two GDX mouse models (Supplementary Figure S5). Moreover, spontaneous SH foci in aged mice were neither enriched in nor derived from Dlk1-expressing cells (Supplementary Figure S6), supporting the hypothesis that SH results from a de-differentiation event [<span>7</span>]. Interestingly, Dlk1 was re-expressed in an autochthonous mouse model of ACC, in which concomitant inactivation of <i>Trp53</i> and activation of <i>Ctnnb1</i>, driven by the aldosterone synthase promoter (<i>BPCre</i>) [<span>8</span>], leads to ACC formation with high penetrance. In 23 tumor samples from 17 mice (9 female), Dlk1 expression was low or absent in benign and pre-malignant tumors, moderate in localized ACC, and higher in metastatic disease, both in the primary tumors and in lung metastases. There was a stepwise increase of Dlk1 expression with disease severity, and a positive correlation between Dlk1 expression and age (Figure 1G-H, Supplementary Figure S7). These results indicate that in the <i>BPCre</i> model, Dlk1, rather than marking the cell of origin, is re-expressed in ACC, potentially conferring cancer stem cell characteristics.</p><p>In a prospective discovery cohort of 73 consecutive patients (26 male) undergoing adrenalectomy in London, UK (Supplementary Table S1), DLK1 expression was significantly higher in ACC than in benign adrenal disease and normal adrenals (Supplementary Figure S8A). This finding was validated in a larger cohort from Würzburg, Germany, comprising 178 ACC tumor samples from 159 patients (53 male) (Supplementary Table S2). DLK1 expression was ubiquitous and heterogenous, with apparent clones of DLK1-positive cells, similar to those observed in <i>BPCre</i> mice. DLK1 expression was not correlated with age, sex, or tumor size and remained constant across different European Network for the Study of Adrenal Tumors (ENS@T) tumor stages, hormonal activity of tumors, Weiss score, and Ki-67% (Supplementary Figure S8B-H). As in <i>BPCre</i> mice, DLK1 expression was present in recurrent human disease and could clearly identify metastases from background tissue. There was a significant positive correlation between DLK1 expression in primary tumors and in recurrent/metastatic disease in the same patients (Figure 1I-J), marking DLK1 expression as a disease defining feature of disease progression.</p><p>In primary ACC (<i>n</i> = 88), higher DLK1 expression was associated with a stepwise increase in the risk of disease recurrence, which remained independently significant in multivariate Cox regression analysis (Figure 1K, Supplementary Table S3, Supplementary Figure S8I-J). In all ACC samples (<i>n</i> = 176), higher DLK1 expression trended toward an increased risk of disease progression, though this did not reach statistical significance in multivariate Cox analysis (<i>P</i> = 0.079) (Supplementary Figure S8K, Supplementary Table S3). However, higher DLK1 expression was significantly associated with an increased risk of progression in ENS@T stage I &amp; II disease (Figure 1L-M). These data suggest the metastatic potential of ACC may be influenced by DLK1 levels. RNA sequencing of the ACC cell line H295R, with DLK1 overexpression and knockdown, revealed that higher DLK1 expression was associated with lower expression of immune signaling gene set, suggesting that the carcinogenic role of DLK1 may, in part, be mediated through mechanisms associated with senescence-induced immune remodeling [<span>9</span>] (Supplementary Figure S9A-E).</p><p>DLK1 has a cleavable ectodomain that is detectable in serum. Serum Dlk1 levels were significantly higher in <i>BPCre</i> mice (compared to age-matched controls) and in two subcutaneous tumor mouse models: one using the <i>BPCre</i> tumor-derived cell line BCH-ACC3A [<span>10</span>] and another injected with H295R cells (Figure 1N-Q). In all cases, there was a strong positive correlation between tumor size and serum DLK1 levels (Supplementary Figure S10). In humans, pre-operative serum DLK1 levels were significantly higher in ACC than in benign adrenocortical adenomas in the London cohort and could predict the diagnosis of ACC with high sensitivity and specificity (Figure 1R-S). This finding was validated in the German cohort, where significantly higher serum DLK1 levels were observed in patients with a greater disease burden (Figure 1T, Supplementary Table S4). As in tissue, serum DLK1 levels did not correlate with other prognostic or clinicopathological features (Supplementary Figure S11A-F). Post-operative blood samples showed a significant reduction in DLK1 levels after tumor resection (Figure 1U). Pre-operative serum DLK1 levels positively correlated with tissue DLK1 expression in both cohorts (Figure 1V, Supplementary Figure S11G). These findings indicate that serum DLK1 is derived from ACC, with levels reflecting the DLK1 expression of the primary tumor and the extent of disease.</p><p>Spatial whole-transcriptome profiling was performed on DLK1<sup>+</sup> and DLK1<sup>−</sup> regions within four human ACCs. Surprisingly, steroid biosynthesis was the gene ontology pathway most enriched in the DLK1<sup>+</sup> group, consistent with the upregulation of cholesterol synthesis genes, suggesting that DLK1<sup>+</sup> areas have higher steroidogenic potential than DLK1<sup>−</sup> areas (Figure 1W, Supplementary Figures S12-S13). This finding was further supported by increased expression of adrenal differentiation genes with higher DLK1 dosage in the H295R transcriptomic data (Supplementary Figure S9F-H). To further investigate this apparent paradox of enhanced steroidogenic potential in ACC cells expressing an adrenocortical stem cell marker, four different human ACC cell lines (H295R, MUC-1, TVBF7 and CU-ACC1) and one mouse ACC cell line (BCH-ACC3A) were cultured as spheroids. DLK1 expression was significantly enhanced in 3D versus 2D culture in H295R, TVBF7, and CU-ACC1, and interestingly, <i>de novo</i> expression of DLK1 protein was observed in MUC-1 (Figure 1X, Supplementary Figure S14A-F). Liquid chromatography with tandem mass spectrometry revealed that 3D spheroids had significantly increased output of steroids compared to 2D cells in H295R, CU-ACC1, and BCH-ACC3A, with a trend toward increased steroidogenesis in MUC-1 and TVBF7 (Supplementary Table S5). Fluorescence-activated cell sorting showed that DLK1<sup>+</sup> cells generated significantly more colony-forming units than DLK1<sup>−</sup> populations after 21 days in culture (Figure 1Y, Supplementary Figure S14G-H). These findings suggest that ACC cells expressing a bona fide adrenocortical stem cell marker possess superior steroidogenic potential while retaining some progenitor cell features, providing a possible explanation for the negative prognostic impact of DLK1 expression in ACC.</p><p>These data define Dlk1 as a novel adrenocortical stem/progenitor cell marker with a role in both adrenocortical organogenesis and malignancy development. Expression data from mice and human ACC indicate that DLK1 is associated with increased malignancy and tumor aggressiveness. Furthermore, DLK1 holds promise as a biomarker for the diagnosis, prognosis, and follow-up of patients with ACC, particularly through serum measurements using a benchtop assay. Further larger prospective studies are needed to confirm this role, along with investigations into DLK1 as a potential therapeutic target in ACC, given its preferential expression in this malignancy.</p><p><i>Conceptualization</i>: Leonardo Guasti, James F.H. Pittaway, and Katia Mariniello. <i>Methodology</i>: Leonardo Guasti, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Irene Hadjidemetriou, Silviu Sbiera, Matthias Kroiss, Martin Fassnacht, William M. Drake, Kleiton Silva Borges, and David T. Breault. <i>Validation</i>: Kleiton Silva Borges, Claudio Ribeiro, Katia Mariniello, James F.H. Pittaway, Barabara Altieri, Jiang A. Lim, David T. Breault, David S. Tourigny and Charlotte Hall. <i>Formal analysis</i>: James F.H. Pittaway, Katia Mariniello, Barbara Altieri, and Kleiton Silva Borges. <i>Investigation</i>: Gerard Ruiz-Babot, Oliver Rayner, David R. Taylor, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Leonardo Guasti, Silviu Sbiera, Carles Gaston-Massuet, and Emanuel Rognoni. <i>Resources</i>: Sandra Sigala, Andrea Abate, Mariangela Tamburello, Katja Kiseljak-Vassiliades, Margaret Wierman, Laila Parvanta, Tarek E. Abdel-Aziz, Teng-Teng Chung, Aimee Di Marco, Fausto Palazzo, Celso E. Gomez-Sanchez, Constanze Hantel, Julie Foster, Julie Cleaver, Jane Sosabowski, Nafis Rahman, Milena Doroszko, and Cristina L. Ronchi. <i>Data curation</i>: James F.H. Pittaway, Katia Mariniello, and Leonardo Guasti. <i>Writing-original draft</i>: James F.H. Pittaway, Katia Mariniello, and Leonardo Guasti. <i>Writing-review and editing</i>: all authors. <i>Supervision</i>: Leonardo Guasti, William M. Drake, Martin Fassnacht, Matthias Kroiss, David T. Breault. <i>Project administration</i>: Leonardo Guasti.</p><p>The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.</p><p>This work was supported by the MRC (MR/X021017/1, MR/S022155/1), BBSRC (BB/V007246/1), Barts Charity (MGU0436), Rosetrees Trust (M355-F1), The Medical College of Saint Bartholomew's Hospital Trust, the German Research Foundation (Deutsche Forschungsgemeinschaft, 314061271), and the National Institutes of Health Physician-Scientist Career Development Award (R01DK123694).</p><p>Human adrenal specimens were collected from patients undergoing surgery at St Bartholomew's, University College and Hammersmith Hospitals, London, after obtaining written informed consent from participants and in accordance with the study protocol Genetics of endocrine tumors (REC: 06/Q0104/133). In Germany, all tissue was collected under the ENS@T research ethical agreement (No. 88/11) at the Universitätsklinikum Würzburg. All patients provided informed consent. All clinical data were collected through the ENS@T database (registry.ensat.org).</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":"45 6","pages":"663-668"},"PeriodicalIF":20.1000,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.70012","citationCount":"0","resultStr":"{\"title\":\"Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma\",\"authors\":\"Katia Mariniello,&nbsp;James F. H. Pittaway,&nbsp;Barbara Altieri,&nbsp;Kleiton Silva Borges,&nbsp;Irene Hadjidemetriou,&nbsp;Claudio Ribeiro,&nbsp;Gerard Ruiz-Babot,&nbsp;David S. Tourigny,&nbsp;Jiang A. Lim,&nbsp;Julie Foster,&nbsp;Julie Cleaver,&nbsp;Jane Sosabowski,&nbsp;Nafis Rahman,&nbsp;Milena Doroszko,&nbsp;Constanze Hantel,&nbsp;Sandra Sigala,&nbsp;Andrea Abate,&nbsp;Mariangela Tamburello,&nbsp;Katja Kiseljak-Vassiliades,&nbsp;Margaret Wierman,&nbsp;Charlotte Hall,&nbsp;Laila Parvanta,&nbsp;Tarek E. Abdel-Aziz,&nbsp;Teng-Teng Chung,&nbsp;Aimee Di Marco,&nbsp;Fausto Palazzo,&nbsp;Celso E. Gomez-Sanchez,&nbsp;David R. Taylor,&nbsp;Oliver Rayner,&nbsp;Cristina L. Ronchi,&nbsp;Carles Gaston-Massuet,&nbsp;Silviu Sbiera,&nbsp;William M. Drake,&nbsp;Emanuel Rognoni,&nbsp;Matthias Kroiss,&nbsp;David T. Breault,&nbsp;Martin Fassnacht,&nbsp;Leonardo Guasti\",\"doi\":\"10.1002/cac2.70012\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Adrenocortical carcinoma (ACC) is a rare malignancy with no widely available biomarkers and commonly presents at later stages with a bleak prognosis [<span>1</span>]. Dysregulation of signaling pathways involved in the organogenesis and homeostasis of the adrenal cortex is implicated in its pathogenesis [<span>2</span>]. The paternally expressed, cleavable protein delta-like non-canonical Notch ligand 1 (DLK1) is expressed in rat adrenocortical progenitor cells [<span>3</span>] and in clusters of relatively undifferentiated cells in the human adrenal gland [<span>4</span>]. Its expression is rare in most adult human tissues but has been reported across various cancers, often associated with worse survival [<span>5</span>]. Here we define the role of DLK1 in adrenocortical development, self-renewal, and the development and progression of ACC.</p><p>Dlk1<sup>+</sup> cells were present in both the capsule and cortex during embryonic development but became restricted to the capsule postnatally in both male and female mice (Supplementary Figure S1), with minimal overlap in expression with Axin-2 (Wnt-active) cells, their early descendants, and platelet-derived growth factor receptor alpha (PDGFRα), a marker of mesenchymal stem/fibroblastic cells (Supplementary Figure S2). Dlk1 cells were rarely positive for Ki-67, whereas <i>Gli1</i> expression in the capsule, unlike Dlk1, remained high during development and throughout postnatal life (Supplementary Figure S3). Genetic lineage tracing using inducible <i>Dlk1<sup>CreERT2/+</sup></i>; <i>Rosa<sup>tdTomato/+</sup></i> mice showed that Dlk1<sup>+</sup> cells functioned as adrenocortical stem cells during development (Figure 1A-F), but were largely dormant postnatally and inactive during postnatal adrenocortical remodeling (Supplementary Figure S4).</p><p>Capsular-like cells are pathognomonic of subcapsular hyperplasia (SH), a histological hallmark in mouse adrenals that occurs spontaneously in aged females and in certain strains/transgenic models after gonadectomy (GDX) [<span>6</span>]. SH foci are thought to represent a morphological continuum progressing toward adrenocortical tumors. Dlk1 was not expressed in SH or in subsequent tumors in two GDX mouse models (Supplementary Figure S5). Moreover, spontaneous SH foci in aged mice were neither enriched in nor derived from Dlk1-expressing cells (Supplementary Figure S6), supporting the hypothesis that SH results from a de-differentiation event [<span>7</span>]. Interestingly, Dlk1 was re-expressed in an autochthonous mouse model of ACC, in which concomitant inactivation of <i>Trp53</i> and activation of <i>Ctnnb1</i>, driven by the aldosterone synthase promoter (<i>BPCre</i>) [<span>8</span>], leads to ACC formation with high penetrance. In 23 tumor samples from 17 mice (9 female), Dlk1 expression was low or absent in benign and pre-malignant tumors, moderate in localized ACC, and higher in metastatic disease, both in the primary tumors and in lung metastases. There was a stepwise increase of Dlk1 expression with disease severity, and a positive correlation between Dlk1 expression and age (Figure 1G-H, Supplementary Figure S7). These results indicate that in the <i>BPCre</i> model, Dlk1, rather than marking the cell of origin, is re-expressed in ACC, potentially conferring cancer stem cell characteristics.</p><p>In a prospective discovery cohort of 73 consecutive patients (26 male) undergoing adrenalectomy in London, UK (Supplementary Table S1), DLK1 expression was significantly higher in ACC than in benign adrenal disease and normal adrenals (Supplementary Figure S8A). This finding was validated in a larger cohort from Würzburg, Germany, comprising 178 ACC tumor samples from 159 patients (53 male) (Supplementary Table S2). DLK1 expression was ubiquitous and heterogenous, with apparent clones of DLK1-positive cells, similar to those observed in <i>BPCre</i> mice. DLK1 expression was not correlated with age, sex, or tumor size and remained constant across different European Network for the Study of Adrenal Tumors (ENS@T) tumor stages, hormonal activity of tumors, Weiss score, and Ki-67% (Supplementary Figure S8B-H). As in <i>BPCre</i> mice, DLK1 expression was present in recurrent human disease and could clearly identify metastases from background tissue. There was a significant positive correlation between DLK1 expression in primary tumors and in recurrent/metastatic disease in the same patients (Figure 1I-J), marking DLK1 expression as a disease defining feature of disease progression.</p><p>In primary ACC (<i>n</i> = 88), higher DLK1 expression was associated with a stepwise increase in the risk of disease recurrence, which remained independently significant in multivariate Cox regression analysis (Figure 1K, Supplementary Table S3, Supplementary Figure S8I-J). In all ACC samples (<i>n</i> = 176), higher DLK1 expression trended toward an increased risk of disease progression, though this did not reach statistical significance in multivariate Cox analysis (<i>P</i> = 0.079) (Supplementary Figure S8K, Supplementary Table S3). However, higher DLK1 expression was significantly associated with an increased risk of progression in ENS@T stage I &amp; II disease (Figure 1L-M). These data suggest the metastatic potential of ACC may be influenced by DLK1 levels. RNA sequencing of the ACC cell line H295R, with DLK1 overexpression and knockdown, revealed that higher DLK1 expression was associated with lower expression of immune signaling gene set, suggesting that the carcinogenic role of DLK1 may, in part, be mediated through mechanisms associated with senescence-induced immune remodeling [<span>9</span>] (Supplementary Figure S9A-E).</p><p>DLK1 has a cleavable ectodomain that is detectable in serum. Serum Dlk1 levels were significantly higher in <i>BPCre</i> mice (compared to age-matched controls) and in two subcutaneous tumor mouse models: one using the <i>BPCre</i> tumor-derived cell line BCH-ACC3A [<span>10</span>] and another injected with H295R cells (Figure 1N-Q). In all cases, there was a strong positive correlation between tumor size and serum DLK1 levels (Supplementary Figure S10). In humans, pre-operative serum DLK1 levels were significantly higher in ACC than in benign adrenocortical adenomas in the London cohort and could predict the diagnosis of ACC with high sensitivity and specificity (Figure 1R-S). This finding was validated in the German cohort, where significantly higher serum DLK1 levels were observed in patients with a greater disease burden (Figure 1T, Supplementary Table S4). As in tissue, serum DLK1 levels did not correlate with other prognostic or clinicopathological features (Supplementary Figure S11A-F). Post-operative blood samples showed a significant reduction in DLK1 levels after tumor resection (Figure 1U). Pre-operative serum DLK1 levels positively correlated with tissue DLK1 expression in both cohorts (Figure 1V, Supplementary Figure S11G). These findings indicate that serum DLK1 is derived from ACC, with levels reflecting the DLK1 expression of the primary tumor and the extent of disease.</p><p>Spatial whole-transcriptome profiling was performed on DLK1<sup>+</sup> and DLK1<sup>−</sup> regions within four human ACCs. Surprisingly, steroid biosynthesis was the gene ontology pathway most enriched in the DLK1<sup>+</sup> group, consistent with the upregulation of cholesterol synthesis genes, suggesting that DLK1<sup>+</sup> areas have higher steroidogenic potential than DLK1<sup>−</sup> areas (Figure 1W, Supplementary Figures S12-S13). This finding was further supported by increased expression of adrenal differentiation genes with higher DLK1 dosage in the H295R transcriptomic data (Supplementary Figure S9F-H). To further investigate this apparent paradox of enhanced steroidogenic potential in ACC cells expressing an adrenocortical stem cell marker, four different human ACC cell lines (H295R, MUC-1, TVBF7 and CU-ACC1) and one mouse ACC cell line (BCH-ACC3A) were cultured as spheroids. DLK1 expression was significantly enhanced in 3D versus 2D culture in H295R, TVBF7, and CU-ACC1, and interestingly, <i>de novo</i> expression of DLK1 protein was observed in MUC-1 (Figure 1X, Supplementary Figure S14A-F). Liquid chromatography with tandem mass spectrometry revealed that 3D spheroids had significantly increased output of steroids compared to 2D cells in H295R, CU-ACC1, and BCH-ACC3A, with a trend toward increased steroidogenesis in MUC-1 and TVBF7 (Supplementary Table S5). Fluorescence-activated cell sorting showed that DLK1<sup>+</sup> cells generated significantly more colony-forming units than DLK1<sup>−</sup> populations after 21 days in culture (Figure 1Y, Supplementary Figure S14G-H). These findings suggest that ACC cells expressing a bona fide adrenocortical stem cell marker possess superior steroidogenic potential while retaining some progenitor cell features, providing a possible explanation for the negative prognostic impact of DLK1 expression in ACC.</p><p>These data define Dlk1 as a novel adrenocortical stem/progenitor cell marker with a role in both adrenocortical organogenesis and malignancy development. Expression data from mice and human ACC indicate that DLK1 is associated with increased malignancy and tumor aggressiveness. Furthermore, DLK1 holds promise as a biomarker for the diagnosis, prognosis, and follow-up of patients with ACC, particularly through serum measurements using a benchtop assay. Further larger prospective studies are needed to confirm this role, along with investigations into DLK1 as a potential therapeutic target in ACC, given its preferential expression in this malignancy.</p><p><i>Conceptualization</i>: Leonardo Guasti, James F.H. Pittaway, and Katia Mariniello. <i>Methodology</i>: Leonardo Guasti, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Irene Hadjidemetriou, Silviu Sbiera, Matthias Kroiss, Martin Fassnacht, William M. Drake, Kleiton Silva Borges, and David T. Breault. <i>Validation</i>: Kleiton Silva Borges, Claudio Ribeiro, Katia Mariniello, James F.H. Pittaway, Barabara Altieri, Jiang A. Lim, David T. Breault, David S. Tourigny and Charlotte Hall. <i>Formal analysis</i>: James F.H. Pittaway, Katia Mariniello, Barbara Altieri, and Kleiton Silva Borges. <i>Investigation</i>: Gerard Ruiz-Babot, Oliver Rayner, David R. Taylor, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Leonardo Guasti, Silviu Sbiera, Carles Gaston-Massuet, and Emanuel Rognoni. <i>Resources</i>: Sandra Sigala, Andrea Abate, Mariangela Tamburello, Katja Kiseljak-Vassiliades, Margaret Wierman, Laila Parvanta, Tarek E. Abdel-Aziz, Teng-Teng Chung, Aimee Di Marco, Fausto Palazzo, Celso E. Gomez-Sanchez, Constanze Hantel, Julie Foster, Julie Cleaver, Jane Sosabowski, Nafis Rahman, Milena Doroszko, and Cristina L. Ronchi. <i>Data curation</i>: James F.H. Pittaway, Katia Mariniello, and Leonardo Guasti. <i>Writing-original draft</i>: James F.H. Pittaway, Katia Mariniello, and Leonardo Guasti. <i>Writing-review and editing</i>: all authors. <i>Supervision</i>: Leonardo Guasti, William M. Drake, Martin Fassnacht, Matthias Kroiss, David T. Breault. <i>Project administration</i>: Leonardo Guasti.</p><p>The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.</p><p>This work was supported by the MRC (MR/X021017/1, MR/S022155/1), BBSRC (BB/V007246/1), Barts Charity (MGU0436), Rosetrees Trust (M355-F1), The Medical College of Saint Bartholomew's Hospital Trust, the German Research Foundation (Deutsche Forschungsgemeinschaft, 314061271), and the National Institutes of Health Physician-Scientist Career Development Award (R01DK123694).</p><p>Human adrenal specimens were collected from patients undergoing surgery at St Bartholomew's, University College and Hammersmith Hospitals, London, after obtaining written informed consent from participants and in accordance with the study protocol Genetics of endocrine tumors (REC: 06/Q0104/133). In Germany, all tissue was collected under the ENS@T research ethical agreement (No. 88/11) at the Universitätsklinikum Würzburg. All patients provided informed consent. All clinical data were collected through the ENS@T database (registry.ensat.org).</p>\",\"PeriodicalId\":9495,\"journal\":{\"name\":\"Cancer Communications\",\"volume\":\"45 6\",\"pages\":\"663-668\"},\"PeriodicalIF\":20.1000,\"publicationDate\":\"2025-03-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.70012\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cancer Communications\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cac2.70012\",\"RegionNum\":1,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ONCOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.70012","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
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摘要

这些数据表明,ACC的转移潜力可能受到DLK1水平的影响。对DLK1过表达和低表达的ACC细胞系H295R的RNA测序显示,DLK1的高表达与免疫信号基因集的低表达相关,表明DLK1的致癌作用可能在一定程度上通过与衰老诱导的免疫重塑[9]相关的机制介导(Supplementary Figure S9A-E)。DLK1具有可切割的外结构域,可在血清中检测到。BPCre小鼠(与年龄匹配的对照组相比)和两种皮下肿瘤小鼠模型的血清Dlk1水平显著升高:一种使用BPCre肿瘤来源细胞系BCH-ACC3A[10],另一种注射H295R细胞(图1N-Q)。在所有病例中,肿瘤大小与血清DLK1水平有很强的正相关(Supplementary Figure S10)。在伦敦队列中,人类ACC患者术前血清DLK1水平明显高于良性肾上腺皮质腺瘤患者,可以以高灵敏度和特异性预测ACC的诊断(图1R-S)。这一发现在德国队列中得到了验证,在疾病负担更重的患者中观察到明显更高的血清DLK1水平(图1T,补充表S4)。与组织中一样,血清DLK1水平与其他预后或临床病理特征无关(补充图S11A-F)。术后血液样本显示肿瘤切除后DLK1水平显著降低(图1U)。两组患者术前血清DLK1水平与组织DLK1表达呈正相关(图1V,补充图S11G)。这些发现表明,血清DLK1来源于ACC,其水平反映了原发肿瘤的DLK1表达和疾病的程度。对四个人类acc的DLK1+和DLK1−区域进行空间全转录组分析。令人惊讶的是,类固醇生物合成是DLK1+组中最富集的基因本体途径,与胆固醇合成基因的上调一致,这表明DLK1+区域比DLK1−区域具有更高的类固醇生成潜力(图1W,补充图S12-S13)。在H295R转录组数据中,随着DLK1剂量的增加,肾上腺分化基因的表达增加,进一步支持了这一发现(补充图S9F-H)。为了进一步研究表达一种肾上腺皮质干细胞标记物的ACC细胞中类固醇生成潜能增强这一明显的悖论,我们将四种不同的人ACC细胞系(H295R、muc1、TVBF7和CU-ACC1)和一种小鼠ACC细胞系(BCH-ACC3A)培养成球体。与2D培养相比,H295R、TVBF7和CU-ACC1在3D培养中DLK1表达显著增强,有趣的是,在MUC-1中观察到DLK1蛋白的从头表达(图1X,补充图S14A-F)。液相色谱串联质谱分析显示,与2D细胞相比,H295R、CU-ACC1和BCH-ACC3A中3D球体的类固醇分泌量明显增加,并且在muc1和TVBF7中有增加类固醇生成的趋势(补充表S5)。荧光激活细胞分选显示,培养21天后,DLK1+细胞产生的集落形成单位明显多于DLK1−群体(图1Y,补充图S14G-H)。这些发现表明,表达真正的肾上腺皮质干细胞标记物的ACC细胞在保留一些祖细胞特征的同时具有优越的类固醇生成潜力,这可能解释了DLK1在ACC中的表达对预后的负面影响。这些数据将Dlk1定义为一种新的肾上腺皮质干细胞/祖细胞标志物,在肾上腺皮质器官发生和恶性肿瘤发展中都有作用。小鼠和人ACC的表达数据表明,DLK1与恶性肿瘤和肿瘤侵袭性增加有关。此外,DLK1有望作为ACC患者的诊断、预后和随访的生物标志物,特别是通过使用台式测定法进行血清测量。考虑到DLK1在这种恶性肿瘤中的优先表达,需要进一步更大规模的前瞻性研究来证实这一作用,并研究DLK1作为ACC的潜在治疗靶点。概念化:Leonardo Guasti, James F.H. Pittaway和Katia Mariniello。研究方法:Leonardo Guasti, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Irene Hadjidemetriou, Silviu Sbiera, Matthias Kroiss, Martin Fassnacht, William M. Drake, Kleiton Silva Borges和David T. breult。验证:Kleiton Silva Borges, Claudio Ribeiro, Katia Mariniello, James F.H. Pittaway, Barabara Altieri, Jiang A. Lim, David T. breult, David S. Tourigny和Charlotte Hall。形式分析:James F.H. Pittaway, Katia Mariniello, Barbara Altieri和Kleiton Silva Borges。调查:Gerard Ruiz-Babot, Oliver Rayner, David R. Taylor, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Leonardo Guasti, Silviu Sbiera, Carles Gaston-Massuet和Emanuel Rognoni。资源:Sandra Sigala, Andrea Abate, Mariangela Tamburello, Katja kiseljk - vassiliades, Margaret Wierman, Laila Parvanta, Tarek E. Abdel-Aziz, tenteng - teng Chung, Aimee Di Marco, Fausto Palazzo, Celso E. Gomez-Sanchez, Constanze Hantel, Julie Foster, Julie Cleaver, Jane Sosabowski, Nafis Rahman, Milena Doroszko和Cristina L. Ronchi。数据管理:James F.H. Pittaway, Katia Mariniello和Leonardo Guasti。原稿:James F.H. Pittaway, Katia Mariniello和Leonardo Guasti。写作-评审和编辑:所有作者。监督:Leonardo Guasti, William M. Drake, Martin Fassnacht, Matthias Kroiss, David T. breult项目管理:Leonardo Guasti。作者声明在本文的研究、作者身份和/或发表方面没有潜在的利益冲突。这项工作得到了MRC (MR/X021017/1, MR/S022155/1), BBSRC (BB/V007246/1), Barts Charity (MGU0436), Rosetrees Trust (M355-F1),圣巴塞洛缪医学院医院信托基金,德国研究基金会(Deutsche Forschungsgemeinschaft, 314061271)和美国国立卫生研究院医师-科学家职业发展奖(R01DK123694)的支持。在获得参与者的书面知情同意后,根据研究方案内分泌肿瘤遗传学(REC: 06/Q0104/133),从伦敦圣巴塞洛缪大学学院和哈默史密斯医院接受手术的患者收集人体肾上腺标本。在德国,所有组织都是根据Universitätsklinikum ww<e:1> rzburg的ENS@T研究伦理协议(No. 88/11)收集的。所有患者均提供知情同意。所有临床数据均通过ENS@T数据库(registry.ensat.org)收集。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma

Dlk1 is a novel adrenocortical stem/progenitor cell marker that predicts malignancy in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare malignancy with no widely available biomarkers and commonly presents at later stages with a bleak prognosis [1]. Dysregulation of signaling pathways involved in the organogenesis and homeostasis of the adrenal cortex is implicated in its pathogenesis [2]. The paternally expressed, cleavable protein delta-like non-canonical Notch ligand 1 (DLK1) is expressed in rat adrenocortical progenitor cells [3] and in clusters of relatively undifferentiated cells in the human adrenal gland [4]. Its expression is rare in most adult human tissues but has been reported across various cancers, often associated with worse survival [5]. Here we define the role of DLK1 in adrenocortical development, self-renewal, and the development and progression of ACC.

Dlk1+ cells were present in both the capsule and cortex during embryonic development but became restricted to the capsule postnatally in both male and female mice (Supplementary Figure S1), with minimal overlap in expression with Axin-2 (Wnt-active) cells, their early descendants, and platelet-derived growth factor receptor alpha (PDGFRα), a marker of mesenchymal stem/fibroblastic cells (Supplementary Figure S2). Dlk1 cells were rarely positive for Ki-67, whereas Gli1 expression in the capsule, unlike Dlk1, remained high during development and throughout postnatal life (Supplementary Figure S3). Genetic lineage tracing using inducible Dlk1CreERT2/+; RosatdTomato/+ mice showed that Dlk1+ cells functioned as adrenocortical stem cells during development (Figure 1A-F), but were largely dormant postnatally and inactive during postnatal adrenocortical remodeling (Supplementary Figure S4).

Capsular-like cells are pathognomonic of subcapsular hyperplasia (SH), a histological hallmark in mouse adrenals that occurs spontaneously in aged females and in certain strains/transgenic models after gonadectomy (GDX) [6]. SH foci are thought to represent a morphological continuum progressing toward adrenocortical tumors. Dlk1 was not expressed in SH or in subsequent tumors in two GDX mouse models (Supplementary Figure S5). Moreover, spontaneous SH foci in aged mice were neither enriched in nor derived from Dlk1-expressing cells (Supplementary Figure S6), supporting the hypothesis that SH results from a de-differentiation event [7]. Interestingly, Dlk1 was re-expressed in an autochthonous mouse model of ACC, in which concomitant inactivation of Trp53 and activation of Ctnnb1, driven by the aldosterone synthase promoter (BPCre) [8], leads to ACC formation with high penetrance. In 23 tumor samples from 17 mice (9 female), Dlk1 expression was low or absent in benign and pre-malignant tumors, moderate in localized ACC, and higher in metastatic disease, both in the primary tumors and in lung metastases. There was a stepwise increase of Dlk1 expression with disease severity, and a positive correlation between Dlk1 expression and age (Figure 1G-H, Supplementary Figure S7). These results indicate that in the BPCre model, Dlk1, rather than marking the cell of origin, is re-expressed in ACC, potentially conferring cancer stem cell characteristics.

In a prospective discovery cohort of 73 consecutive patients (26 male) undergoing adrenalectomy in London, UK (Supplementary Table S1), DLK1 expression was significantly higher in ACC than in benign adrenal disease and normal adrenals (Supplementary Figure S8A). This finding was validated in a larger cohort from Würzburg, Germany, comprising 178 ACC tumor samples from 159 patients (53 male) (Supplementary Table S2). DLK1 expression was ubiquitous and heterogenous, with apparent clones of DLK1-positive cells, similar to those observed in BPCre mice. DLK1 expression was not correlated with age, sex, or tumor size and remained constant across different European Network for the Study of Adrenal Tumors (ENS@T) tumor stages, hormonal activity of tumors, Weiss score, and Ki-67% (Supplementary Figure S8B-H). As in BPCre mice, DLK1 expression was present in recurrent human disease and could clearly identify metastases from background tissue. There was a significant positive correlation between DLK1 expression in primary tumors and in recurrent/metastatic disease in the same patients (Figure 1I-J), marking DLK1 expression as a disease defining feature of disease progression.

In primary ACC (n = 88), higher DLK1 expression was associated with a stepwise increase in the risk of disease recurrence, which remained independently significant in multivariate Cox regression analysis (Figure 1K, Supplementary Table S3, Supplementary Figure S8I-J). In all ACC samples (n = 176), higher DLK1 expression trended toward an increased risk of disease progression, though this did not reach statistical significance in multivariate Cox analysis (P = 0.079) (Supplementary Figure S8K, Supplementary Table S3). However, higher DLK1 expression was significantly associated with an increased risk of progression in ENS@T stage I & II disease (Figure 1L-M). These data suggest the metastatic potential of ACC may be influenced by DLK1 levels. RNA sequencing of the ACC cell line H295R, with DLK1 overexpression and knockdown, revealed that higher DLK1 expression was associated with lower expression of immune signaling gene set, suggesting that the carcinogenic role of DLK1 may, in part, be mediated through mechanisms associated with senescence-induced immune remodeling [9] (Supplementary Figure S9A-E).

DLK1 has a cleavable ectodomain that is detectable in serum. Serum Dlk1 levels were significantly higher in BPCre mice (compared to age-matched controls) and in two subcutaneous tumor mouse models: one using the BPCre tumor-derived cell line BCH-ACC3A [10] and another injected with H295R cells (Figure 1N-Q). In all cases, there was a strong positive correlation between tumor size and serum DLK1 levels (Supplementary Figure S10). In humans, pre-operative serum DLK1 levels were significantly higher in ACC than in benign adrenocortical adenomas in the London cohort and could predict the diagnosis of ACC with high sensitivity and specificity (Figure 1R-S). This finding was validated in the German cohort, where significantly higher serum DLK1 levels were observed in patients with a greater disease burden (Figure 1T, Supplementary Table S4). As in tissue, serum DLK1 levels did not correlate with other prognostic or clinicopathological features (Supplementary Figure S11A-F). Post-operative blood samples showed a significant reduction in DLK1 levels after tumor resection (Figure 1U). Pre-operative serum DLK1 levels positively correlated with tissue DLK1 expression in both cohorts (Figure 1V, Supplementary Figure S11G). These findings indicate that serum DLK1 is derived from ACC, with levels reflecting the DLK1 expression of the primary tumor and the extent of disease.

Spatial whole-transcriptome profiling was performed on DLK1+ and DLK1 regions within four human ACCs. Surprisingly, steroid biosynthesis was the gene ontology pathway most enriched in the DLK1+ group, consistent with the upregulation of cholesterol synthesis genes, suggesting that DLK1+ areas have higher steroidogenic potential than DLK1 areas (Figure 1W, Supplementary Figures S12-S13). This finding was further supported by increased expression of adrenal differentiation genes with higher DLK1 dosage in the H295R transcriptomic data (Supplementary Figure S9F-H). To further investigate this apparent paradox of enhanced steroidogenic potential in ACC cells expressing an adrenocortical stem cell marker, four different human ACC cell lines (H295R, MUC-1, TVBF7 and CU-ACC1) and one mouse ACC cell line (BCH-ACC3A) were cultured as spheroids. DLK1 expression was significantly enhanced in 3D versus 2D culture in H295R, TVBF7, and CU-ACC1, and interestingly, de novo expression of DLK1 protein was observed in MUC-1 (Figure 1X, Supplementary Figure S14A-F). Liquid chromatography with tandem mass spectrometry revealed that 3D spheroids had significantly increased output of steroids compared to 2D cells in H295R, CU-ACC1, and BCH-ACC3A, with a trend toward increased steroidogenesis in MUC-1 and TVBF7 (Supplementary Table S5). Fluorescence-activated cell sorting showed that DLK1+ cells generated significantly more colony-forming units than DLK1 populations after 21 days in culture (Figure 1Y, Supplementary Figure S14G-H). These findings suggest that ACC cells expressing a bona fide adrenocortical stem cell marker possess superior steroidogenic potential while retaining some progenitor cell features, providing a possible explanation for the negative prognostic impact of DLK1 expression in ACC.

These data define Dlk1 as a novel adrenocortical stem/progenitor cell marker with a role in both adrenocortical organogenesis and malignancy development. Expression data from mice and human ACC indicate that DLK1 is associated with increased malignancy and tumor aggressiveness. Furthermore, DLK1 holds promise as a biomarker for the diagnosis, prognosis, and follow-up of patients with ACC, particularly through serum measurements using a benchtop assay. Further larger prospective studies are needed to confirm this role, along with investigations into DLK1 as a potential therapeutic target in ACC, given its preferential expression in this malignancy.

Conceptualization: Leonardo Guasti, James F.H. Pittaway, and Katia Mariniello. Methodology: Leonardo Guasti, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Irene Hadjidemetriou, Silviu Sbiera, Matthias Kroiss, Martin Fassnacht, William M. Drake, Kleiton Silva Borges, and David T. Breault. Validation: Kleiton Silva Borges, Claudio Ribeiro, Katia Mariniello, James F.H. Pittaway, Barabara Altieri, Jiang A. Lim, David T. Breault, David S. Tourigny and Charlotte Hall. Formal analysis: James F.H. Pittaway, Katia Mariniello, Barbara Altieri, and Kleiton Silva Borges. Investigation: Gerard Ruiz-Babot, Oliver Rayner, David R. Taylor, James F.H. Pittaway, Katia Mariniello, Barbara Altieri, Leonardo Guasti, Silviu Sbiera, Carles Gaston-Massuet, and Emanuel Rognoni. Resources: Sandra Sigala, Andrea Abate, Mariangela Tamburello, Katja Kiseljak-Vassiliades, Margaret Wierman, Laila Parvanta, Tarek E. Abdel-Aziz, Teng-Teng Chung, Aimee Di Marco, Fausto Palazzo, Celso E. Gomez-Sanchez, Constanze Hantel, Julie Foster, Julie Cleaver, Jane Sosabowski, Nafis Rahman, Milena Doroszko, and Cristina L. Ronchi. Data curation: James F.H. Pittaway, Katia Mariniello, and Leonardo Guasti. Writing-original draft: James F.H. Pittaway, Katia Mariniello, and Leonardo Guasti. Writing-review and editing: all authors. Supervision: Leonardo Guasti, William M. Drake, Martin Fassnacht, Matthias Kroiss, David T. Breault. Project administration: Leonardo Guasti.

The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.

This work was supported by the MRC (MR/X021017/1, MR/S022155/1), BBSRC (BB/V007246/1), Barts Charity (MGU0436), Rosetrees Trust (M355-F1), The Medical College of Saint Bartholomew's Hospital Trust, the German Research Foundation (Deutsche Forschungsgemeinschaft, 314061271), and the National Institutes of Health Physician-Scientist Career Development Award (R01DK123694).

Human adrenal specimens were collected from patients undergoing surgery at St Bartholomew's, University College and Hammersmith Hospitals, London, after obtaining written informed consent from participants and in accordance with the study protocol Genetics of endocrine tumors (REC: 06/Q0104/133). In Germany, all tissue was collected under the ENS@T research ethical agreement (No. 88/11) at the Universitätsklinikum Würzburg. All patients provided informed consent. All clinical data were collected through the ENS@T database (registry.ensat.org).

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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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