MOLECULAR & CELLULAR ONCOLOGY 2016, VOL. 3, NO. 3, e1168508 (3 pages) http://dx.doi.org/10.1080/23723556.2016.1168508
AUTHOR’S VIEW
Transcriptome profiling links the intrinsic properties of human prostate basal cells to prostate cancer aggressiveness Dingxiao Zhanga,b a Department of Epigenetics and Molecular Carcinogenesis, the University of Texas MD Anderson Cancer Center, Smithville, TX, USA; bDepartment of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY
ABSTRACT
ARTICLE HISTORY
Genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations reveals the unexpected findings that basal cells preferentially express gene categories associated with stem cells, neurogenesis, and rRNA biogenesis. Importantly, the basal cell gene expression profile is enriched in clinically advanced, anaplastic, castration-resistant, and metastatic prostate cancers.
Received 14 March 2016 Revised 15 March 2016 Accepted 16 March 2016 KEYWORDS
Basal stem cells; human prostate epithelial cells; neurogenesis; prostate cancer; transcriptome
Prostate cancer (PCa) is a heterogeneous malignancy harboring phenotypically and functionally diverse subpopulations of cancer cells.1 To better understand PCa cell heterogeneity, it is crucial to dissect the biology of normal prostate epithelial lineages to provide a foundation for discoveries that can impact our understanding and treatment of the disease. The prostate is an exocrine gland in which prostatic ducts are lined by 3 cell types: secretory luminal cells, basal cells, and rare neuroendocrine cells.2 Developmentally, the murine prostate originates from an ancestral p63C basal stem cell (SC) population.3 In the human prostate there is also evidence that the basal cell layer harbors regenerative SCs.4 Gene expression is a key determinant of cellular phenotypes. An in-depth understanding of gene expression differences between basal and luminal cells in normal human prostate will help illuminate intrinsic functional differences between these 2 cell types, which in turn could offer fresh insights into the cell of origin for different types of PCa. Here, we highlight our recent deep RNA-sequencing (RNA-seq) analysis that defined the genome-wide transcriptomes of freshly purified benign human prostatic basal and luminal cell populations.5 Our deep RNA-seq analysis revealed that human prostatic basal and luminal cells have distinctly different transcriptomes.5 Basal cells preferentially express gene signatures similar to those enriched in embryonic and mammary SCs, consistent with basal cells acting as undifferentiated stem-like cells. Luminal cells express gene signatures more closely resembling differentiated, functional cell types. For example, basal cells preferentially express developmental and SC genes such as SHH, HMGA2, SOX2, MYC, and genes involved in WNT, NOTCH, and FGF/IGF/TGFb signaling. Furthermore, the gene expression patterns indicate that basal and luminal cells reciprocally regulate each other in both autocrine and paracrine fashions (Fig. 1A).5 For example, basal cells preferentially CONTACT Dingxiao Zhang © 2016 Taylor & Francis Group, LLC
[email protected]
express multiple NOTCH ligands and receptors, whereas luminal cells specifically express the NOTCH ligand DLL4. Likewise, basal cells express FGFR3/4 and several FGFR ligands, whereas luminal cells highly express only the ligand FGF13. Surprisingly, basal cells express both WNT ligands and inhibitors, suggesting a self-regulated balance of WNT signaling. Collectively, these findings suggest that basal cells are primarily autocrine regulated but receive paracrine signals from neighboring luminal cells (Fig. 1A), consistent with basal cell SC properties such as self-sustenance.6 Strikingly, we found that more than 11% of genes upregulated in basal cells have neurogenic roles such as promoting neural development, axonal guidance, and neural progenitor functions. Consistent with this finding, primary basal cells can undergo spontaneous or induced “neural” development in vitro, generating neural stem cell (NSC)-like cells. This result is consistent with the notion that the neural lineage is the “default” cell fate for embryonic stem cells (ESCs) when they undergo spontaneous differentiation.7 In addition to crosstalk between the basal and luminal epithelial layers, our data also indicates an extensive cross-communication between epithelial cells and the underlying extracellular matrix and stroma components (Fig. 1B). Surprisingly, approximately 7.5% of the luminal cell-specific genes are also proneural genes but, in contrast to neurogenic roles, these genes promote neuronal signal reception and processing. Considering that the prostate is a richly innervated organ8 and the prostate stroma has readily detectable cells that express neural/neuronal markers such as GFAP, TH, NES, and b-tubulin III (Fig. 1B), we speculate that luminal cells, and perhaps some basal cells, constantly communicate with the stromal nervous system to rapidly respond to microenvironmental changes. Thus, this neural signal-enriched stroma might establish a SC niche for the basal SCs that endows them with proneural capacities.
e1168508-2
D. ZHANG
Figure 1. Possible crosstalk between prostatic epithelial cells and their microenvironment. (A) Transcriptome-derived models for crosstalk of representative signaling pathways between prostatic basal and luminal cells (see text for detail). (B) Potential crosstalk between epithelial cells and the extracellular matrix and stromal cells. Many different types of cells, including neural/neuronal-like cells, reside in the stroma and may functionally contribute to the cellular plasticity of basal cells. (C) Basal cells molecularly resemble anaplastic prostate cancer (PCa) and castration-resistant prostate cancer (CRPC), suggesting that they may function directly as the cells of origin for these aggressive variants and/or indirectly as the cells of origin for adenocarcinomas through differentiation into luminal cells. ADT, androgen deprivation therapy.
One of our most significant findings is the link between the basal gene expression profile and aggressive PCa subtypes and adverse patient outcomes. Specifically, the basal cell gene expression profile resembles the profiles of high-grade primary tumors and anaplastic PCa (including various undifferentiated variants, such as small cell carcinoma, large-cell neuroendocrine carcinoma, and neuroendocrine PCa).5,9 Anaplastic PCa occurs in fewer than 5% of PCa patients; however, repeat biopsies show that this increases to 10–20% during castration resistant PCa (CRPC) progression. Notably, the basal cell gene expression profile is enriched for CRPC gene signatures, which are associated with metastasis and poor patient survival.5 Similarly, a recent report indicated that human metastatic breast cancer cells express a basal/stem-like transcriptional program.10 We suggest that the basal cell gene expression profile might be developed as a “biomarker” for aggressive PCa with poor prognosis. As we look toward the future, we propose that the molecular resemblance of basal cell gene expression to that of anaplastic PCa and CRPC provides an advance in our understanding of these diverse, poorly characterized, and aggressive PCa subtypes.5 In principle, genes that are critically important for maintaining basal cell stemness may also operate in anaplastic and CRPC cells and thus constitute therapeutic targets. Intriguingly, besides the SC and neurogenesis-related genes, basal cells also overexpress MYC and its transcriptional program as well as genes required for rRNA biogenesis (Fig. 1C). MYC is known to promote rRNA gene expression, and both overexpression of MYC and increased transcription of rRNA genes are common features of human cancer. This connection may suggest a rationale for treating anaplastic PCa and CRPC with polymerase I (Pol I) inhibition. Indeed, our preliminary evidence shows that the Pol I inhibitor CX-5461 demonstrates
significant therapeutic efficacy against experimental models of CRPC (unpublished data). Together, our results reinforce the concept that MYC and the MYC-mediated transcriptional program represent critical therapeutic targets in aggressive PCa.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References 1. Liu X, Chen X, Rycaj K, Chao HP, Deng Q, Jeter C, Liu C, Honorio S, Li H, Davis T, et al. Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells. Oncotarget 2015; 6:23959-86; PMID:26246472; http://dx.doi.org/10.18632/ oncotarget.4260 2. Shen MM, Abate-Shen C. Molecular genetics of prostate cancer: new prospects for old challenges. Genes Dev 2010; 24:1967-2000; PMID:20844012; http://dx.doi.org/10.1101/gad.1965810 3. Pignon JC, Grisanzio C, Geng Y, Song J, Shivdasani RA, Signoretti S. p63-expressing cells are the stem cells of developing prostate, bladder, and colorectal epithelia. Proc Natl Acad Sci U S A 2013; 110:8105-10; PMID:23620512; http://dx.doi.org/10.1073/pnas.1221216110 4. Collins AT, Habib FK, Maitland NJ, Neal DE. Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)integrin expression. J Cell Sci 2001; 114:3865-72; PMID:11719553 5. Zhang D, Park D, Zhong Y, Lu Y, Rycaj K, Gong S, Chen X, Liu X, Chao HP, Whitney P, et al. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer. Nat Commun 2016; 7:10798; PMID:26924072; http://dx.doi.org/10.1038/ ncomms10798 6. Choi N, Zhang B, Zhang L, Ittmann M, Xin L. Adult murine prostate basal and luminal cells are self-sustained lineages that can both serve as targets for prostate cancer initiation. Cancer Cell 2012; 21:253-65; PMID:22340597; http://dx.doi.org/10.1016/j.ccr.2012.01.005 7. Kamiya D, Banno S, Sasai N, Ohgushi M, Inomata H, Watanabe K, Kawada M, Yakura R, Kiyonari H, Nakao K, et al. Intrinsic transition of
MOLECULAR & CELLULAR ONCOLOGY
embryonic stem-cell differentiation into neural progenitors. Nature 2011; 470:503-9; PMID:21326203; http://dx.doi.org/10.1038/nature09726 8. Magnon C, Hall SJ, Lin J, Xue X, Gerber L, Freedland SJ, Frenette PS. Autonomic nerve development contributes to prostate cancer progression. Science 2013; 341:1236361; PMID:23846904; http://dx.doi.org/ 10.1126/science.1236361 9. Beltran H, Tomlins S, Aparicio A, Arora V, Rickman D, Ayala G, Huang J, True L, Gleave ME, Soule H, et al. Aggressive variants
e1168508-3
of castration-resistant prostate cancer. Clin Cancer Res 2014; 20:2846-50; PMID:24727321; http://dx.doi.org/10.1158/1078-0432. CCR-13-3309 10. Lawson DA, Bhakta NR, Kessenbrock K, Prummel KD, Yu Y, Takai K, Zhou A, Eyob H, Balakrishnan S, Wang CY, et al. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 2015; 526:131-5; PMID:26416748; http://dx.doi.org/10.1038/ nature15260
AUTHOR’S VIEW
Transcriptome profiling links the intrinsic properties of human prostate basal cells to prostate cancer aggressiveness Dingxiao Zhanga,b a Department of Epigenetics and Molecular Carcinogenesis, the University of Texas MD Anderson Cancer Center, Smithville, TX, USA; bDepartment of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY
ABSTRACT
ARTICLE HISTORY
Genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations reveals the unexpected findings that basal cells preferentially express gene categories associated with stem cells, neurogenesis, and rRNA biogenesis. Importantly, the basal cell gene expression profile is enriched in clinically advanced, anaplastic, castration-resistant, and metastatic prostate cancers.
Received 14 March 2016 Revised 15 March 2016 Accepted 16 March 2016 KEYWORDS
Basal stem cells; human prostate epithelial cells; neurogenesis; prostate cancer; transcriptome
Prostate cancer (PCa) is a heterogeneous malignancy harboring phenotypically and functionally diverse subpopulations of cancer cells.1 To better understand PCa cell heterogeneity, it is crucial to dissect the biology of normal prostate epithelial lineages to provide a foundation for discoveries that can impact our understanding and treatment of the disease. The prostate is an exocrine gland in which prostatic ducts are lined by 3 cell types: secretory luminal cells, basal cells, and rare neuroendocrine cells.2 Developmentally, the murine prostate originates from an ancestral p63C basal stem cell (SC) population.3 In the human prostate there is also evidence that the basal cell layer harbors regenerative SCs.4 Gene expression is a key determinant of cellular phenotypes. An in-depth understanding of gene expression differences between basal and luminal cells in normal human prostate will help illuminate intrinsic functional differences between these 2 cell types, which in turn could offer fresh insights into the cell of origin for different types of PCa. Here, we highlight our recent deep RNA-sequencing (RNA-seq) analysis that defined the genome-wide transcriptomes of freshly purified benign human prostatic basal and luminal cell populations.5 Our deep RNA-seq analysis revealed that human prostatic basal and luminal cells have distinctly different transcriptomes.5 Basal cells preferentially express gene signatures similar to those enriched in embryonic and mammary SCs, consistent with basal cells acting as undifferentiated stem-like cells. Luminal cells express gene signatures more closely resembling differentiated, functional cell types. For example, basal cells preferentially express developmental and SC genes such as SHH, HMGA2, SOX2, MYC, and genes involved in WNT, NOTCH, and FGF/IGF/TGFb signaling. Furthermore, the gene expression patterns indicate that basal and luminal cells reciprocally regulate each other in both autocrine and paracrine fashions (Fig. 1A).5 For example, basal cells preferentially CONTACT Dingxiao Zhang © 2016 Taylor & Francis Group, LLC
[email protected]
express multiple NOTCH ligands and receptors, whereas luminal cells specifically express the NOTCH ligand DLL4. Likewise, basal cells express FGFR3/4 and several FGFR ligands, whereas luminal cells highly express only the ligand FGF13. Surprisingly, basal cells express both WNT ligands and inhibitors, suggesting a self-regulated balance of WNT signaling. Collectively, these findings suggest that basal cells are primarily autocrine regulated but receive paracrine signals from neighboring luminal cells (Fig. 1A), consistent with basal cell SC properties such as self-sustenance.6 Strikingly, we found that more than 11% of genes upregulated in basal cells have neurogenic roles such as promoting neural development, axonal guidance, and neural progenitor functions. Consistent with this finding, primary basal cells can undergo spontaneous or induced “neural” development in vitro, generating neural stem cell (NSC)-like cells. This result is consistent with the notion that the neural lineage is the “default” cell fate for embryonic stem cells (ESCs) when they undergo spontaneous differentiation.7 In addition to crosstalk between the basal and luminal epithelial layers, our data also indicates an extensive cross-communication between epithelial cells and the underlying extracellular matrix and stroma components (Fig. 1B). Surprisingly, approximately 7.5% of the luminal cell-specific genes are also proneural genes but, in contrast to neurogenic roles, these genes promote neuronal signal reception and processing. Considering that the prostate is a richly innervated organ8 and the prostate stroma has readily detectable cells that express neural/neuronal markers such as GFAP, TH, NES, and b-tubulin III (Fig. 1B), we speculate that luminal cells, and perhaps some basal cells, constantly communicate with the stromal nervous system to rapidly respond to microenvironmental changes. Thus, this neural signal-enriched stroma might establish a SC niche for the basal SCs that endows them with proneural capacities.
e1168508-2
D. ZHANG
Figure 1. Possible crosstalk between prostatic epithelial cells and their microenvironment. (A) Transcriptome-derived models for crosstalk of representative signaling pathways between prostatic basal and luminal cells (see text for detail). (B) Potential crosstalk between epithelial cells and the extracellular matrix and stromal cells. Many different types of cells, including neural/neuronal-like cells, reside in the stroma and may functionally contribute to the cellular plasticity of basal cells. (C) Basal cells molecularly resemble anaplastic prostate cancer (PCa) and castration-resistant prostate cancer (CRPC), suggesting that they may function directly as the cells of origin for these aggressive variants and/or indirectly as the cells of origin for adenocarcinomas through differentiation into luminal cells. ADT, androgen deprivation therapy.
One of our most significant findings is the link between the basal gene expression profile and aggressive PCa subtypes and adverse patient outcomes. Specifically, the basal cell gene expression profile resembles the profiles of high-grade primary tumors and anaplastic PCa (including various undifferentiated variants, such as small cell carcinoma, large-cell neuroendocrine carcinoma, and neuroendocrine PCa).5,9 Anaplastic PCa occurs in fewer than 5% of PCa patients; however, repeat biopsies show that this increases to 10–20% during castration resistant PCa (CRPC) progression. Notably, the basal cell gene expression profile is enriched for CRPC gene signatures, which are associated with metastasis and poor patient survival.5 Similarly, a recent report indicated that human metastatic breast cancer cells express a basal/stem-like transcriptional program.10 We suggest that the basal cell gene expression profile might be developed as a “biomarker” for aggressive PCa with poor prognosis. As we look toward the future, we propose that the molecular resemblance of basal cell gene expression to that of anaplastic PCa and CRPC provides an advance in our understanding of these diverse, poorly characterized, and aggressive PCa subtypes.5 In principle, genes that are critically important for maintaining basal cell stemness may also operate in anaplastic and CRPC cells and thus constitute therapeutic targets. Intriguingly, besides the SC and neurogenesis-related genes, basal cells also overexpress MYC and its transcriptional program as well as genes required for rRNA biogenesis (Fig. 1C). MYC is known to promote rRNA gene expression, and both overexpression of MYC and increased transcription of rRNA genes are common features of human cancer. This connection may suggest a rationale for treating anaplastic PCa and CRPC with polymerase I (Pol I) inhibition. Indeed, our preliminary evidence shows that the Pol I inhibitor CX-5461 demonstrates
significant therapeutic efficacy against experimental models of CRPC (unpublished data). Together, our results reinforce the concept that MYC and the MYC-mediated transcriptional program represent critical therapeutic targets in aggressive PCa.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References 1. Liu X, Chen X, Rycaj K, Chao HP, Deng Q, Jeter C, Liu C, Honorio S, Li H, Davis T, et al. Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells. Oncotarget 2015; 6:23959-86; PMID:26246472; http://dx.doi.org/10.18632/ oncotarget.4260 2. Shen MM, Abate-Shen C. Molecular genetics of prostate cancer: new prospects for old challenges. Genes Dev 2010; 24:1967-2000; PMID:20844012; http://dx.doi.org/10.1101/gad.1965810 3. Pignon JC, Grisanzio C, Geng Y, Song J, Shivdasani RA, Signoretti S. p63-expressing cells are the stem cells of developing prostate, bladder, and colorectal epithelia. Proc Natl Acad Sci U S A 2013; 110:8105-10; PMID:23620512; http://dx.doi.org/10.1073/pnas.1221216110 4. Collins AT, Habib FK, Maitland NJ, Neal DE. Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(1)integrin expression. J Cell Sci 2001; 114:3865-72; PMID:11719553 5. Zhang D, Park D, Zhong Y, Lu Y, Rycaj K, Gong S, Chen X, Liu X, Chao HP, Whitney P, et al. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer. Nat Commun 2016; 7:10798; PMID:26924072; http://dx.doi.org/10.1038/ ncomms10798 6. Choi N, Zhang B, Zhang L, Ittmann M, Xin L. Adult murine prostate basal and luminal cells are self-sustained lineages that can both serve as targets for prostate cancer initiation. Cancer Cell 2012; 21:253-65; PMID:22340597; http://dx.doi.org/10.1016/j.ccr.2012.01.005 7. Kamiya D, Banno S, Sasai N, Ohgushi M, Inomata H, Watanabe K, Kawada M, Yakura R, Kiyonari H, Nakao K, et al. Intrinsic transition of
MOLECULAR & CELLULAR ONCOLOGY
embryonic stem-cell differentiation into neural progenitors. Nature 2011; 470:503-9; PMID:21326203; http://dx.doi.org/10.1038/nature09726 8. Magnon C, Hall SJ, Lin J, Xue X, Gerber L, Freedland SJ, Frenette PS. Autonomic nerve development contributes to prostate cancer progression. Science 2013; 341:1236361; PMID:23846904; http://dx.doi.org/ 10.1126/science.1236361 9. Beltran H, Tomlins S, Aparicio A, Arora V, Rickman D, Ayala G, Huang J, True L, Gleave ME, Soule H, et al. Aggressive variants
e1168508-3
of castration-resistant prostate cancer. Clin Cancer Res 2014; 20:2846-50; PMID:24727321; http://dx.doi.org/10.1158/1078-0432. CCR-13-3309 10. Lawson DA, Bhakta NR, Kessenbrock K, Prummel KD, Yu Y, Takai K, Zhou A, Eyob H, Balakrishnan S, Wang CY, et al. Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 2015; 526:131-5; PMID:26416748; http://dx.doi.org/10.1038/ nature15260
