Low CD38 Identifies Progenitor-like Inflammation-Associated Luminal Cells that Can Initiate Human Prostate Cancer and Predict Poor Outcome

doi:10.1016/j.celrep.2016.11.010

. 2016 Dec 6;17(10):2596-2606.

doi: 10.1016/j.celrep.2016.11.010.

Low CD38 Identifies Progenitor-like Inflammation-Associated Luminal Cells that Can Initiate Human Prostate Cancer and Predict Poor Outcome

Xian Liu¹, Tristan R Grogan², Haley Hieronymus³, Takao Hashimoto¹, Jack Mottahedeh¹, Donghui Cheng⁴, Lijun Zhang⁵, Kevin Huang⁵, Tanya Stoyanova⁶, Jung Wook Park⁶, Ruzanna O Shkhyan¹, Behdokht Nowroozizadeh⁷, Matthew B Rettig⁸, Charles L Sawyers³, David Elashoff⁹, Steve Horvath¹⁰, Jiaoti Huang¹¹, Owen N Witte¹², Andrew S Goldstein¹³

Affiliations

¹ Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Department of Medicine Statistics Core, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Programs in Human Oncology and Pathogenesis, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
⁴ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Division of Hematology-Oncology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; VA Greater Los Angeles Healthcare System, Los Angeles, CA 90024, USA; Urology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Department of Medicine Statistics Core, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁰ Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Biostatistics, UCLA Fielding School of Public Health, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹³ Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Urology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: agoldstein@mednet.ucla.edu.

PMID: 27926864
PMCID: PMC5367888
DOI: 10.1016/j.celrep.2016.11.010

Low CD38 Identifies Progenitor-like Inflammation-Associated Luminal Cells that Can Initiate Human Prostate Cancer and Predict Poor Outcome

Xian Liu et al. Cell Rep. 2016.

. 2016 Dec 6;17(10):2596-2606.

doi: 10.1016/j.celrep.2016.11.010.

Authors

Affiliations

¹ Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Department of Medicine Statistics Core, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Programs in Human Oncology and Pathogenesis, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
⁴ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁶ Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁷ Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁸ Division of Hematology-Oncology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; VA Greater Los Angeles Healthcare System, Los Angeles, CA 90024, USA; Urology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Department of Medicine Statistics Core, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁰ Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Biostatistics, UCLA Fielding School of Public Health, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹² Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA; Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹³ Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Urology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: agoldstein@mednet.ucla.edu.

PMID: 27926864
PMCID: PMC5367888
DOI: 10.1016/j.celrep.2016.11.010

Abstract

Inflammation is a risk factor for prostate cancer, but the mechanisms by which inflammation increases that risk are poorly understood. Here, we demonstrate that low expression of CD38 identifies a progenitor-like subset of luminal cells in the human prostate. CD38^lo luminal cells are enriched in glands adjacent to inflammatory cells and exhibit epithelial nuclear factor κB (NF-κB) signaling. In response to oncogenic transformation, CD38^lo luminal cells can initiate human prostate cancer in an in vivo tissue-regeneration assay. Finally, the CD38^lo luminal phenotype and gene signature are associated with disease progression and poor outcome in prostate cancer. Our results suggest that prostate inflammation expands the pool of progenitor-like target cells susceptible to tumorigenesis.

Keywords: CD38; cell of origin; inflammation; luminal; progenitor; prostate cancer; stem cells; transformation.

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Figures

**Figure 1. Differential Expression of CD38 Enables Isolation of Discrete Luminal Subsets from Fresh Human Prostate Tissue**
(A) Fluorescence-activated cell sorting analysis of dissociated benign human prostate cells demonstrates CD38 expression in EpCAM⁺ CD45⁻ CD26⁺ luminal cells, but not in EpCAM⁺ CD45⁻ CD49f-hi basal cells. (B) Immunohistochemical analysis of benign human prostate glands stained with anti-CD38 antibodies. Boxed region is magnified in the right. Arrows identify rare CD38^lo cells lining the gland. Scale bars, 50 μm. (C) Fluorescence-activated cell sorting analysis of dissociated benign human prostate cells gated on EpCAM⁺ CD45⁻ cells and further stained with antibodies against CD49f and CD38. (D) Intracellular flow cytometry for Keratin 18 (K18) and surface staining for CD38 and CD49f. Gating on K18⁺ luminal cells demonstrates two luminal populations in right plot. (E) Western blot analysis of three epithelial subsets from representative patient sample stained with antibodies against CD38, AR, p63, keratin 14 (K14), and histone H3 as a loading control. (F) Sorted cells were counted in a hemocytometer, and average cell diameter (in microns) was quantified for each population from multiple patient samples. Data represent mean ± SEM with replicate points shown. Newman-Keuls multiple comparison test with ***p < 0.0001.

**Figure 2. CD38^lo Cells Exhibit Greater Progenitor Activity than CD38^hi Luminal Cells In Vitro**
(A) Flow cytometry analysis for CD49f and CD38 dissociated human prostate epithelium as gated during the sort (left), and post-sort analysis of CD38^lo and CD38^hi luminal cells prior to in vitro culture. (B and C) Primary human benign prostate epithelial cells (EpCAM⁺ CD45⁻) were sorted into basal cells, CD38^lo and CD38^hi luminal subsets and plated for in vitro colony and sphere forming assays. Results from in vitro two-dimensional colony-forming assay (B) and three-dimensional sphere-forming assay (C) measured 10–14 days after plating benign human prostate epithelial subsets. Data represent pooled experiments from three independent patients. Mean ± SEM is shown. Statistics represent Newman-Keuls multiple comparison test with *p < 0.05, ***p < 0.0005. Representative images of outgrowths from each epithelial subset are taken at 10–14 days. Scale bars, 250 μm. (D) CD38^lo luminal cells generate significantly more organoids than CD38^hi luminal cells, four replicates per sample, representative of five independent patient samples. Statistics represent Two-tailed unpaired t test with ****p < 0.0001. (E) Representative staining of CD38^lo luminal cell-derived organoids for K5, K8, and DAPI nuclear counterstain. Scale bars, 50 μm. (F) Representative images of organoids forming from single CD38^lo luminal cells taken at day 1 and day 10. Scale bars, 100 μm.

**Figure 3. CD38^lo Luminal Cells Are Associated with Inflammation**
(A) Heatmap of RNA-sequencing results of CD38^lo luminal cells and CD38^hi luminal cells from three independent patients. (B) DAVID bioinformatics analysis (SP_PIR_ KEYWORDS) of the CD38^lo luminal gene signature with log(10) p values plotted. (C) Western blot analysis of CD38^lo and CD38^hi luminal cells from a representative benign human prostate sample stained for total p65, phosphorylated p65 (P-p65 S536), BCL2, and histone H3 as a loading control. (D) Immunohistochemical analysis of benign human prostate stained for CD38 and phosphorylated p65 (S536) on serial sections indicating an inverse relationship between expression of CD38 and P-p65. Scale bars, 50 μm. (E) Immunohistochemical analysis of benign human prostate glands stained with anti-CD38 antibodies. Boxed regions are magnified in the bottom row. Arrows denote regions of epithelium with low expression of CD38 in close proximity to red asterisk identifying areas with immune cell infiltration. Scale bars, 100 μm. (F) Immunohistochemistry on serial slides stained for CD38 and CD45. Scale bars, 100 μm. (G) Increased proportion of luminal cells with CD38^lo phenotype from 29 samples plotted as CD45⁺ cells less than or greater than 7% of total prostate. Statistics represent two-tailed t test; ***p = 0.0003.

**Figure 4. CD38^lo Luminal Cells Exhibit Reduced Androgen Signaling**
(A) Relative expression of *Klk3* or K8 compared to *Gapdh* in basal cells as measured by qPCR of basal, CD38^lo, and CD38^hi luminal cells isolated from three independent patient samples. Data are presented as mean ± SEM with replicate points shown. *Klk3* is shown on a log scale. Two-tailed unpaired t test between CD38^lo and CD38^hi luminal subsets; ***p = 0.0004. (B) Immunohistochemical analysis of benign human prostate stained for CD38 and AR targets PSA, FKBP5, and MSMB. Scale bars, 100 μm. (C) Immunohistochemistry for CD38, PSA, and AR distinguishes CD38^hi and CD38^lo regions. AR expression is magnified to demonstrate reduced expression in CD38^lo cells. Scale bars, 50 μm.

**Figure 5. Association of CD38^lo Phenotype with Prostate Cancer Progression**
(A) Plot of composite CD38 staining score in tissues histologically classified as normal, prostate intra-epithelial neoplasia (PIN), or prostate cancer (CA) with Gleason scores less than 7 or 7–10. Middle bar represents mean value. The overall effect of different levels of CD38 among the prostate regions was confirmed (p < 0.001). (B) Representative immunohistochemical images of normal, PIN, Gleason 6, and Gleason 8 cancer stained for CD38, with high-power images below. Scale bars, 50 μm. (C and D) Survival analysis from MSKCC cohort measuring biochemical recurrence (C) and metastasis (D) for tumors containing CD38 mRNA 1 SD below the mean compared to the remainder (non-CD38^lo). Log rank (Mantel Cox) p < 0.001 (recurrence in C) and p = 0.002 (metastasis in D). (E) Survival analysis from the TCGA cohort measuring biochemical recurrence comparing CD38 expression less than (CD38^lo) or greater than (non- CD38^lo) the mean Z score. Log rank (Mantel Cox) p = 0.001.

**Figure 6. PSCA Is Expressed in CD38^lo Luminal Cells**
(A) Serial sections of benign human prostate stained for CD38 and PSCA with boxed regions to magnify distinct CD38^hi PSCA-lo glands and inflammation-associated CD38^lo PSCA-hi cells. (B) Serial sections of human prostate tissue stained for CD38 and PSCA with boxed region magnifying CD38^hi PSCA-lo benign tissue separated by a dotted line from CD38^lo PSCA-hi cancer. Scale bars, 50 μm.

**Figure 7. CD38^lo Luminal Cells Can Generate Glands and Initiate Human Prostate Cancer In Vivo**
(A) Schematic representing experimental flow where CD38^lo luminal cells are transduced with lentivirus, expanded in vitro 7–10 days, combined with UGSM and Matrigel and implanted in vivo. Regenerated tissues from representative experiments are shown on the right. Scale bars, 5 mm. (B) Representative in vivo regeneration of CD38^lo luminal cell-derived organoids transduced with control fluorescent GFP and red fluorescent protein (RFP) stained for H&E, K5, K8, DAPI nuclear counterstain, AR, and p63. Scale bars, 50 μm. (C) Representative adenocarcinoma in regenerated tumor tissues from CD38^lo luminal cells transduced with Myc, AKT, and AR stained for H&E, K5, K8, DAPI nuclear counterstain, AR, p63, Ki67, PSA, PSCA, and CD38. Scale bars, 25 μm.

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Comment in

Prostate cancer: On the down-low - low luminal cell CD38 expression is prognostic.
Stone L. Stone L. Nat Rev Urol. 2017 Mar;14(3):133. doi: 10.1038/nrurol.2016.269. Epub 2017 Jan 10. Nat Rev Urol. 2017. PMID: 28072402 No abstract available.
Re: Low CD38 Identifies Progenitor-Like Inflammation-Associated Luminal Cells that Can Initiate Human Prostate Cancer and Predict Poor Outcome.
Atala A. Atala A. J Urol. 2017 Jul;198(1):103. doi: 10.1016/j.juro.2017.04.060. Epub 2017 Apr 12. J Urol. 2017. PMID: 28618670 No abstract available.

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References

1. Ammirante M, Luo JL, Grivennikov S, Nedospasov S, Karin M. B-cell-derived lymphotoxin promotes castration-resistant prostate cancer. Nature. 2010;464:302–305. - PMC - PubMed
1. Birbach A, Eisenbarth D, Kozakowski N, Ladenhauf E, Schmidt-Supprian M, Schmid JA. Persistent inflammation leads to proliferative neoplasia and loss of smooth muscle cells in a prostate tumor model. Neoplasia. 2011;13:692–703. - PMC - PubMed
1. 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;21:253–265. - PMC - PubMed
1. Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420:860–867. - PMC - PubMed
1. Das Roy L, Pathangey LB, Tinder TL, Schettini JL, Gruber HE, Mukherjee P. Breast-cancer-associated metastasis is significantly increased in a model of autoimmune arthritis. Breast Cancer Res. 2009;11:R56. - PMC - PubMed

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[1] Ammirante M, Luo JL, Grivennikov S, Nedospasov S, Karin M. B-cell-derived lymphotoxin promotes castration-resistant prostate cancer. Nature. 2010;464:302–305. - PMC - PubMed

[2] Ammirante M, Luo JL, Grivennikov S, Nedospasov S, Karin M. B-cell-derived lymphotoxin promotes castration-resistant prostate cancer. Nature. 2010;464:302–305. - PMC - PubMed

[3] Birbach A, Eisenbarth D, Kozakowski N, Ladenhauf E, Schmidt-Supprian M, Schmid JA. Persistent inflammation leads to proliferative neoplasia and loss of smooth muscle cells in a prostate tumor model. Neoplasia. 2011;13:692–703. - PMC - PubMed

[4] Birbach A, Eisenbarth D, Kozakowski N, Ladenhauf E, Schmidt-Supprian M, Schmid JA. Persistent inflammation leads to proliferative neoplasia and loss of smooth muscle cells in a prostate tumor model. Neoplasia. 2011;13:692–703. - PMC - PubMed

[5] 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;21:253–265. - PMC - PubMed

[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;21:253–265. - PMC - PubMed

[7] Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420:860–867. - PMC - PubMed

[8] Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420:860–867. - PMC - PubMed

[9] Das Roy L, Pathangey LB, Tinder TL, Schettini JL, Gruber HE, Mukherjee P. Breast-cancer-associated metastasis is significantly increased in a model of autoimmune arthritis. Breast Cancer Res. 2009;11:R56. - PMC - PubMed

[10] Das Roy L, Pathangey LB, Tinder TL, Schettini JL, Gruber HE, Mukherjee P. Breast-cancer-associated metastasis is significantly increased in a model of autoimmune arthritis. Breast Cancer Res. 2009;11:R56. - PMC - PubMed

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Low CD38 Identifies Progenitor-like Inflammation-Associated Luminal Cells that Can Initiate Human Prostate Cancer and Predict Poor Outcome

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Low CD38 Identifies Progenitor-like Inflammation-Associated Luminal Cells that Can Initiate Human Prostate Cancer and Predict Poor Outcome

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