- Email: [email protected]
Genomic Aberrations Drive Clonal Evolution of Neuroendocrine Tumors
TEM 1123 No. of Pages 3
Spotlight
Genomic Aberrations Drive Clonal Evolution of Neuroendocrine Tumors Akash Kumar Kaushik1 and Arun Sreekumar1,2,3,* Molecular features of castrationresistant neuroendocrine prostate cancer (CRPC-NE) are not well characterized. A recent study that investigated genomic aberrations of CRPC-NE tumors suggests their clonal evolution from CRPC adenocarcinoma. Furthermore, the existence of a distinct DNA methylation profile in CRPC-NE implicates a critical role for epigenetic modification in the development of CRPC-NE. Resistance to androgen deprivation therapy (ADT), which aims to target androgen receptor (AR) signaling in metastatic prostate adenocarcinoma, is the root cause of lethal prostate cancer. Recently, a growing body of evidence has implied the emergence of AR-independent CRPCNE tumors as one of the leading causes of resistance to ADT [1]. Moreover, the scarcity of reliable biomarkers to identify patients who may develop CRPC-NE has made it challenging to appropriately prioritize therapeutic interventions. A major hindrance to the accurate diagnosis of CRPC-NE is our lack of understanding of the dynamic molecular changes associated with CRPC-NE and how they evolve during ADT. Remarkable work done by Beltran et al., published in Nature Medicine [2], has provided an in-depth look into the genomic modifications associated with CRPC-NE and castration-resistant adenocarcinoma prostate cancer (CRPC-Adeno),
which may lead to the development of reliable biomarkers for CRPC-NE. In their report, the authors have employed hypothesis-driven models with an eye towards answering the following fundamental questions: (i) What are the critical global genomic aberrations that distinguish CRPC-NE from CRPC-Adeno tumors? (ii) How do CRPCNE tumors evolve during the course of ADT? (iii) Can genomic features serve as reliable biomarkers to determine patients at high risk of developing CRPC-NE tumors? Results of this landmark investigation provide a nearly complete picture of the molecular underpinnings of CRPC-NE. The authors’ findings suggest an adaptation of AR-positive CRPC adenocarcinoma to AR-independent CRPC neuroendocrine tumors following therapies targeting AR signaling. To address these questions, tumor samples were collected from a broad range of metastatic sites from patients with primary adenocarcinoma, CRPC-Adeno, and CRPC-NE. Histological examination of tissues confirmed lower AR protein levels with concomitant downregulation of the mRNA of AR-regulated genes in CRPCNE compared with CRPC-Adeno. To further elucidate the role of additional genomic modifications in CRPC-NE, whole-exome sequencing (WES) was performed on paired 114 metastatic and normal tissues. Interestingly, investigation of WES data identified a significant overlap of genomic aberrations between CRPC-NE and CRPC-Adeno tumors and validated previously identified genomic alterations in CRPC [3,4]. Notably, percentage of concurrent loss of tumor suppressors RB1 (retinoblastoma) and TP53 (p53 tumor suppressor protein), reported to be associated with neuroendocrine-like small cell carcinoma [5], was significantly (P<0.0004) higher in CRPC-NE (53.3%) compared with CRPC-Adeno (13.7%) tissues. Furthermore, careful examination of genomic domains of AR in CRPC-Adeno tumors identified numerous somatic mutations, amplification, and elevated expression of
AR splice variants, such as AR-V7, which is clinically associated with enzalutamide and abiraterone acetate resistance [6]. These AR-associated genomic alterations, however, were almost absent in CRPC-NE tumors, suggesting their reliance on ARindependent mechanisms for growth and proliferation. In addition to defining the distinctive genomic aberrations associated with CRPCNE tumors, a primary goal of this study was to provide molecular evidence regarding the evolution of CRPC-NE. Beltran et al. provide compelling evidence that supports the plausibility of divergent clonal evolution of CRPC-NE tumors from CRPC-Adeno under stress caused by ADT (Figure 1). Correspondingly, consistent and persistent genomic aberrations including deletion/mutation of tumor suppressor genes (BRAC2 and TP53) were detected in tumor samples collected from temporal cases of patients with CRPCNE. These patients were diagnosed with either localized or metastatic adenocarcinoma, and after ADT, patients developed either CRPC-Adeno or CRPC-NE. The presence of such common genomic changes across multiple tumor samples from the same patient suggests divergent clonal evolution of CRPC-NE from AR-positive CRPC adenocarcinoma. Moreover, the model of divergent clonal selection also supports the evident and continuous overlap of mutational and genomic aberrations in adenocarcinoma, CRPC-Adeno, and CRPC-NE. Although informative, these genomic aberrations do not explain the aggressive phenotype of CRPC-NE. To address this, the authors investigated the role of genome-wide epigenetic modification by DNA methylation profiling of CpG islands in tumor samples. Interestingly, DNA methylation, which is involved in the regulation of gene expression, significantly distinguished CRPC-NE from CRPC-Adeno tumors. Furthermore, significant concordance was observed between methylation pattern and transcript level expression of the top differentially altered genes. Moreover, pathway
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
1
TEM 1123 No. of Pages 3
Model for divergent clonal evoluon
∗
∗
DNA
∗ CRPC-Adeno
DNA
∗
CRPC-Adeno
∗
DNA
ADT
+ CRPC-NE
Figure 1. Model for Divergent Clonal Evolution of Castration-Resistant Neuroendocrine Prostate Cancer (CRPC-NE) from Castration-Resistant Adenocarcinoma Prostate Cancer (CRPC-Adeno) Tumors. Tumors resistant to androgen deprivation therapy (ADT) contain unique genomic alterations (purple asterisk, *) as well as those that they share with treatment naïve tumors (black asterisk, *). In addition, CRPC-NE tumors also contain distinct epigenetic modifications (red cross, +) in their genomic DNA.
enrichment analysis using differentially methylated genes in CRPC-NE tumors nominated dysregulation of biological pathways previously reported to play an important role in the biology of NE tumors. For example, expression of EZH2 (a DNA methyltransferase and important regulator of DNA methylation) was observed to be elevated and its downstream typically repressed targets were significantly decreased in both CRPC-NE tumors and corresponding cell line models. Interestingly, as a proof of concept, treatment of neuroendocrine-like cells with an inhibitor of EZH2 significantly decreased their viability compared with adenocarcinomalike cells. Together, these data suggest a critical role for DNA methylation profile in
2
driving the initiation and behavior of tumors. Moreover, the 70-gene signature stood out alone across multiple datasets CRPC-NE tumors. when compared with other established Given the lack of reliable molecular bio- biomarkers. Most importantly, the NEPC markers for the assessment of neuroen- signature also identified neuroendocrine docrine tumors, information from features in tumors defined as adenocarcigenomic, epigenomic, or transcriptomic noma based on histopathological examidata was integrated to determine a 70- nation. Retrospectively, these specific gene neuroendocrine prostate cancer adenocarcinoma tumors failed to respond classifier (NEPC). The robustness of the to ADT with nominal changes in the levels classifier was significantly higher and of serum PSA (prostate-specific antigen, accurate when compared with other reli- biomarker for AR-positive tumors), sugable molecular features the authors tested gesting their reliance on neuroendocrine (e.g., chromogranin A, neuron-specific cell signaling. enolase, synaptophysin). The NEPC signature was further tested in four indepen- In aggregate, this study uncovers a wide dent datasets [4,7–9] and significantly spectrum of molecular aberrations includsegregated CRPC-NE from CRPC-Adeno ing epigenetic reprogramming associated
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
TEM 1123 No. of Pages 3
with CRPC-NE tumors and suggests their provided new areas for therapeutic develdivergent clonal evolution from CRPC- opment for this deadly disease. Adeno tumors. This is consistent with a recent report that demonstrates AR silenc- Acknowledgments ing via DNA methylation as a hallmark of The authors acknowledge Dr Ganesh Palapattu, Chief of Urology Oncology, University of Michigan for his AR-negative tumors, which was reversed critical review and scientific input. using methyltransferase inhibitors [10]. Importantly, these findings provide evi- 1Verna and Marrs McLean Department of Biochemistry dence for clinically evaluating DNA methyl- and Molecular Biology and Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX transferase inhibitors to resensitize CRPC- 77030, USA NE tumors to antiandrogens. Furthermore, 2Department of Molecular and Cellular Biology and Alkek the remarkable robustness of the NEPC Center for Molecular Discovery, Baylor College of Medicine, Houston, TX 77030, USA signature in distinguishing CRPC-NE 3Dan L Duncan Cancer Center, Baylor College of tumors may have potential clinical use as Medicine, Houston, TX 77030, USA predictive markers[1_TD$IF] to effectively guide ther*Correspondence: [email protected] (A. Sreekumar). apeutic decisions. While needing further http://dx.doi.org/10.1016/j.tem.2016.03.009 investigation and corroboration, this work has taken an important step towards References improving our understanding of the global 1. Watson, P.A. et al. (2015) Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. genomic and epigenomic landscapes Nat. Rev. Cancer 15, 701–711 associated with CRPC-NE and further
2. Beltran, H. et al. (2016) Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer. Nat. Med. 22, 298–305 3. Grasso, C.S. et al. (2012) The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239–243 4. Robinson, D. et al. (2015) Integrative clinical genomics of advanced prostate cancer. Cell 161, 1215–1228 5. Zhou, Z. et al. (2006) Synergy of p53 and Rb deficiency in a conditional mouse model for metastatic prostate cancer. Cancer Res. 66, 7889–7898 6. Antonarakis, E.S. et al. (2014) AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N. Engl. J. Med. 371, 1028–1038 7. Beltran, H. et al. (2011) Molecular characterization of neuroendocrine prostate cancer and identification of new drug targets. Cancer Discov. 1, 487–495 8. Cancer Genome Atlas Research Network (2015) The molecular taxonomy of primary prostate cancer. Cell 163, 1011–1025 9. Chakravarty, D. et al. (2014) The oestrogen receptor alpharegulated lncRNA NEAT1 is a critical modulator of prostate cancer. Nat. Commun. 5, 5383 10. Kleb, B. et al. (2016) Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas. Epigenetics Published online February 18, 2016. http://dx.doi.org/10.1080/15592294.2016.1146851
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
3
Spotlight
Genomic Aberrations Drive Clonal Evolution of Neuroendocrine Tumors Akash Kumar Kaushik1 and Arun Sreekumar1,2,3,* Molecular features of castrationresistant neuroendocrine prostate cancer (CRPC-NE) are not well characterized. A recent study that investigated genomic aberrations of CRPC-NE tumors suggests their clonal evolution from CRPC adenocarcinoma. Furthermore, the existence of a distinct DNA methylation profile in CRPC-NE implicates a critical role for epigenetic modification in the development of CRPC-NE. Resistance to androgen deprivation therapy (ADT), which aims to target androgen receptor (AR) signaling in metastatic prostate adenocarcinoma, is the root cause of lethal prostate cancer. Recently, a growing body of evidence has implied the emergence of AR-independent CRPCNE tumors as one of the leading causes of resistance to ADT [1]. Moreover, the scarcity of reliable biomarkers to identify patients who may develop CRPC-NE has made it challenging to appropriately prioritize therapeutic interventions. A major hindrance to the accurate diagnosis of CRPC-NE is our lack of understanding of the dynamic molecular changes associated with CRPC-NE and how they evolve during ADT. Remarkable work done by Beltran et al., published in Nature Medicine [2], has provided an in-depth look into the genomic modifications associated with CRPC-NE and castration-resistant adenocarcinoma prostate cancer (CRPC-Adeno),
which may lead to the development of reliable biomarkers for CRPC-NE. In their report, the authors have employed hypothesis-driven models with an eye towards answering the following fundamental questions: (i) What are the critical global genomic aberrations that distinguish CRPC-NE from CRPC-Adeno tumors? (ii) How do CRPCNE tumors evolve during the course of ADT? (iii) Can genomic features serve as reliable biomarkers to determine patients at high risk of developing CRPC-NE tumors? Results of this landmark investigation provide a nearly complete picture of the molecular underpinnings of CRPC-NE. The authors’ findings suggest an adaptation of AR-positive CRPC adenocarcinoma to AR-independent CRPC neuroendocrine tumors following therapies targeting AR signaling. To address these questions, tumor samples were collected from a broad range of metastatic sites from patients with primary adenocarcinoma, CRPC-Adeno, and CRPC-NE. Histological examination of tissues confirmed lower AR protein levels with concomitant downregulation of the mRNA of AR-regulated genes in CRPCNE compared with CRPC-Adeno. To further elucidate the role of additional genomic modifications in CRPC-NE, whole-exome sequencing (WES) was performed on paired 114 metastatic and normal tissues. Interestingly, investigation of WES data identified a significant overlap of genomic aberrations between CRPC-NE and CRPC-Adeno tumors and validated previously identified genomic alterations in CRPC [3,4]. Notably, percentage of concurrent loss of tumor suppressors RB1 (retinoblastoma) and TP53 (p53 tumor suppressor protein), reported to be associated with neuroendocrine-like small cell carcinoma [5], was significantly (P<0.0004) higher in CRPC-NE (53.3%) compared with CRPC-Adeno (13.7%) tissues. Furthermore, careful examination of genomic domains of AR in CRPC-Adeno tumors identified numerous somatic mutations, amplification, and elevated expression of
AR splice variants, such as AR-V7, which is clinically associated with enzalutamide and abiraterone acetate resistance [6]. These AR-associated genomic alterations, however, were almost absent in CRPC-NE tumors, suggesting their reliance on ARindependent mechanisms for growth and proliferation. In addition to defining the distinctive genomic aberrations associated with CRPCNE tumors, a primary goal of this study was to provide molecular evidence regarding the evolution of CRPC-NE. Beltran et al. provide compelling evidence that supports the plausibility of divergent clonal evolution of CRPC-NE tumors from CRPC-Adeno under stress caused by ADT (Figure 1). Correspondingly, consistent and persistent genomic aberrations including deletion/mutation of tumor suppressor genes (BRAC2 and TP53) were detected in tumor samples collected from temporal cases of patients with CRPCNE. These patients were diagnosed with either localized or metastatic adenocarcinoma, and after ADT, patients developed either CRPC-Adeno or CRPC-NE. The presence of such common genomic changes across multiple tumor samples from the same patient suggests divergent clonal evolution of CRPC-NE from AR-positive CRPC adenocarcinoma. Moreover, the model of divergent clonal selection also supports the evident and continuous overlap of mutational and genomic aberrations in adenocarcinoma, CRPC-Adeno, and CRPC-NE. Although informative, these genomic aberrations do not explain the aggressive phenotype of CRPC-NE. To address this, the authors investigated the role of genome-wide epigenetic modification by DNA methylation profiling of CpG islands in tumor samples. Interestingly, DNA methylation, which is involved in the regulation of gene expression, significantly distinguished CRPC-NE from CRPC-Adeno tumors. Furthermore, significant concordance was observed between methylation pattern and transcript level expression of the top differentially altered genes. Moreover, pathway
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
1
TEM 1123 No. of Pages 3
Model for divergent clonal evoluon
∗
∗
DNA
∗ CRPC-Adeno
DNA
∗
CRPC-Adeno
∗
DNA
ADT
+ CRPC-NE
Figure 1. Model for Divergent Clonal Evolution of Castration-Resistant Neuroendocrine Prostate Cancer (CRPC-NE) from Castration-Resistant Adenocarcinoma Prostate Cancer (CRPC-Adeno) Tumors. Tumors resistant to androgen deprivation therapy (ADT) contain unique genomic alterations (purple asterisk, *) as well as those that they share with treatment naïve tumors (black asterisk, *). In addition, CRPC-NE tumors also contain distinct epigenetic modifications (red cross, +) in their genomic DNA.
enrichment analysis using differentially methylated genes in CRPC-NE tumors nominated dysregulation of biological pathways previously reported to play an important role in the biology of NE tumors. For example, expression of EZH2 (a DNA methyltransferase and important regulator of DNA methylation) was observed to be elevated and its downstream typically repressed targets were significantly decreased in both CRPC-NE tumors and corresponding cell line models. Interestingly, as a proof of concept, treatment of neuroendocrine-like cells with an inhibitor of EZH2 significantly decreased their viability compared with adenocarcinomalike cells. Together, these data suggest a critical role for DNA methylation profile in
2
driving the initiation and behavior of tumors. Moreover, the 70-gene signature stood out alone across multiple datasets CRPC-NE tumors. when compared with other established Given the lack of reliable molecular bio- biomarkers. Most importantly, the NEPC markers for the assessment of neuroen- signature also identified neuroendocrine docrine tumors, information from features in tumors defined as adenocarcigenomic, epigenomic, or transcriptomic noma based on histopathological examidata was integrated to determine a 70- nation. Retrospectively, these specific gene neuroendocrine prostate cancer adenocarcinoma tumors failed to respond classifier (NEPC). The robustness of the to ADT with nominal changes in the levels classifier was significantly higher and of serum PSA (prostate-specific antigen, accurate when compared with other reli- biomarker for AR-positive tumors), sugable molecular features the authors tested gesting their reliance on neuroendocrine (e.g., chromogranin A, neuron-specific cell signaling. enolase, synaptophysin). The NEPC signature was further tested in four indepen- In aggregate, this study uncovers a wide dent datasets [4,7–9] and significantly spectrum of molecular aberrations includsegregated CRPC-NE from CRPC-Adeno ing epigenetic reprogramming associated
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
TEM 1123 No. of Pages 3
with CRPC-NE tumors and suggests their provided new areas for therapeutic develdivergent clonal evolution from CRPC- opment for this deadly disease. Adeno tumors. This is consistent with a recent report that demonstrates AR silenc- Acknowledgments ing via DNA methylation as a hallmark of The authors acknowledge Dr Ganesh Palapattu, Chief of Urology Oncology, University of Michigan for his AR-negative tumors, which was reversed critical review and scientific input. using methyltransferase inhibitors [10]. Importantly, these findings provide evi- 1Verna and Marrs McLean Department of Biochemistry dence for clinically evaluating DNA methyl- and Molecular Biology and Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX transferase inhibitors to resensitize CRPC- 77030, USA NE tumors to antiandrogens. Furthermore, 2Department of Molecular and Cellular Biology and Alkek the remarkable robustness of the NEPC Center for Molecular Discovery, Baylor College of Medicine, Houston, TX 77030, USA signature in distinguishing CRPC-NE 3Dan L Duncan Cancer Center, Baylor College of tumors may have potential clinical use as Medicine, Houston, TX 77030, USA predictive markers[1_TD$IF] to effectively guide ther*Correspondence: [email protected] (A. Sreekumar). apeutic decisions. While needing further http://dx.doi.org/10.1016/j.tem.2016.03.009 investigation and corroboration, this work has taken an important step towards References improving our understanding of the global 1. Watson, P.A. et al. (2015) Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. genomic and epigenomic landscapes Nat. Rev. Cancer 15, 701–711 associated with CRPC-NE and further
2. Beltran, H. et al. (2016) Divergent clonal evolution of castration-resistant neuroendocrine prostate cancer. Nat. Med. 22, 298–305 3. Grasso, C.S. et al. (2012) The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239–243 4. Robinson, D. et al. (2015) Integrative clinical genomics of advanced prostate cancer. Cell 161, 1215–1228 5. Zhou, Z. et al. (2006) Synergy of p53 and Rb deficiency in a conditional mouse model for metastatic prostate cancer. Cancer Res. 66, 7889–7898 6. Antonarakis, E.S. et al. (2014) AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N. Engl. J. Med. 371, 1028–1038 7. Beltran, H. et al. (2011) Molecular characterization of neuroendocrine prostate cancer and identification of new drug targets. Cancer Discov. 1, 487–495 8. Cancer Genome Atlas Research Network (2015) The molecular taxonomy of primary prostate cancer. Cell 163, 1011–1025 9. Chakravarty, D. et al. (2014) The oestrogen receptor alpharegulated lncRNA NEAT1 is a critical modulator of prostate cancer. Nat. Commun. 5, 5383 10. Kleb, B. et al. (2016) Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas. Epigenetics Published online February 18, 2016. http://dx.doi.org/10.1080/15592294.2016.1146851
Trends in Endocrinology & Metabolism, Month Year, Vol. xx, No. yy
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