RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance

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Science  18 Sep 2015:
Vol. 349, Issue 6254, pp. 1351-1356
DOI: 10.1126/science.aab0917

Circulating signals of drug resistance

Cancer drugs often lose their effectiveness because tumors acquire genetic changes that confer drug resistance. Ideally, patients would be switched to a different drug before tumor growth resumes, but this requires early knowledge of how resistance arose. Miyamoto et al. have developed a non-invasive method to spot resistance by sequencing RNA transcripts in single circulating tumor cells (CTCs) (see the Perspective by Nanus and Giannakakou). For example, in prostate cancer patients, drug resistance was triggered by activation of the Wnt signaling pathway. But CTCs are rare and fragile, and the technology needs further development before it is used in clinical practice.

Science, this issue p. 1351; see also p. 1283


Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

After the initial response of metastatic prostate cancer to androgen deprivation therapy (ADT), it invariably recurs as castration-resistant disease (1). Second-line inhibitors of the androgen receptor (AR) have been shown to increase overall survival in castration-resistant prostate cancer (CRPC), consistent with the reactivation of AR signaling in the tumor, but responses are heterogeneous and often short-lived, and resistance to therapy is a pressing clinical problem (1). In other types of cancer, molecular analyses of serial biopsies have enabled the study of acquired drug-resistance mechanisms, intratumor heterogeneity, and tumor evolution in response to therapy (2)—an approach that is restricted by the predominance of bone metastases in prostate cancer (3, 4). Thus, isolation of circulating tumor cells (CTCs) may enable noninvasive monitoring, as patients initially respond and subsequently become refractory to therapies targeting the AR pathway (5). Here, we established single-cell RNA-sequencing (RNA-Seq) profiles of CTCs, individually isolated after microfluidic enrichment from blood specimens of men with prostate cancer, to address their heterogeneity within and across different patients and their differences from primary tumor specimens. Retrospective analyses of clinical and molecular data were then performed to identify potentially clinically relevant mechanisms of acquired drug resistance.

Building on earlier approaches for capturing and scoring CTCs (3), highly efficient microfluidic technologies enable molecular analyses (69). We applied the CTC-iChip to magnetically deplete normal hematopoietic cells from whole-blood specimens (10). Untagged and unfixed CTCs were identified by cell surface staining for epithelial and mesenchymal markers [epithelial cell adhesion molecule (EpCAM) and cadherin-11 (CDH11), respectively], and absent staining for the common leukocyte marker CD45. These labeled CTCs were then individually micromanipulated (fig. S1, A and B). A total of 221 single-candidate prostate CTCs were isolated from 18 patients with metastatic prostate cancer and 4 patients with localized prostate cancer (fig. S1C and table S1). Of these, 133 cells (60%) had RNA of sufficient quality for amplification and next-generation RNA sequencing, and 122 (55%) had >100,000 uniquely aligned sequencing reads (11) (figs. S1C and S2A). Although many cancer cells in the circulation appear to undergo apoptosis, the presence of intact RNA identifies the subset enriched for viable cells. In addition to candidate CTCs, we also obtained comprehensive transcriptomes for bulk primary prostate cancers from a separate cohort of 12 patients (macrodissected for >70% tumor content) (table S2), 30 single cells derived from four different prostate cancer cell lines, and five patient-derived leukocyte controls (fig. S1C). The leukocytes were readily distinguished by their expression of hematopoietic lineage markers and served to exclude any CTCs with potentially contaminating signals. Strict expression thresholds were used to define lineage-confirmed CTCs, scored by prostate lineage-specific genes (PSA, PSMA, AMACR, and AR) and standard epithelial markers (KRT7, KRT8, KRT18, KRT19, and EPCAM) (11) (fig. S2B). Given the presence of leukocyte transcripts suggestive of cellular contamination or misidentification during selection, 28 cells were excluded, and, given low expression of both prostate lineage-specific genes and standard epithelial markers, 17 cells were excluded. The remaining 77 cells (from 13 patients; average of six CTCs per patient) were defined as categorical CTCs (fig. S1C and table S1).

Unsupervised hierarchical clustering analysis of single prostate CTCs, primary tumor samples, and cancer cell lines resulted in their organization into distinct clusters (Fig. 1A). Single CTCs from an individual patient showed considerably greater intercellular heterogeneity in their transcriptional profiles than single cells from prostate cancer cell lines (Fig. 1, B and C) (mean correlation coefficient 0.10 versus 0.44, P < 1 × 10−20), but they strongly clustered according to patient of origin, which indicated higher diversity in CTCs from different patients (Fig. 1C and fig. S2C) (mean correlation coefficient 0.10 for CTCs within patient versus 0.0014 for CTCs between patients, P = 2.0 × 10−11).

Fig. 1 Single-cell RNA-sequencing of prostate CTCs.

(A) Heat map of unsupervised hierarchical clustering analysis of RNA-sequencing data from 77 single lineage–confirmed prostate CTCs, 12 primary tumor samples, and 30 single cells from four prostate cancer cell lines. (B) Heterogeneity, measured by mean correlation coefficient within individual samples with three or more cells available for analysis. (C) Heterogeneity analysis showing mean correlation coefficients from expression data for CTCs between and within patients (0.0013838 versus 0.10055; Holm corrected P = 2.0 × 10−11), and for prostate cancer cell lines between and within lines (0.11568 versus 0.43534; Holm corrected P = 5.42 × 10−14).

We examined gene markers of prostate lineage, epithelial, mesenchymal, and stem cell fates, and cellular proliferation (Fig. 2A). Epithelial markers were abundantly expressed [>10 reads per million (rpm)] by nearly all CTCs analyzed (92%), whereas mesenchymal genes were not up-regulated compared with primary tumors or prostate cancer–derived cell lines. Among robustly expressed transcripts were putative stem cell markers (12), including ALDH7A1, CD44, and KLF4, present in 60% of CTCs. In addition, 47% of CTCs expressed markers of cell proliferation. We performed differential gene expression analysis to identify genes that are up-regulated in prostate CTCs compared with primary tumor samples. A total of 711 genes were highly expressed in CTCs compared with primary tumors; the most enriched were (i) the molecular chaperone HSP90AA1, which regulates the activation and stability of AR, among other functions (13), and (ii) the noncoding RNA transcript MALAT1, which has been implicated in alternative mRNA splicing and transcriptional control of gene expression (14) (Fig. 2B, fig. S4A, and table S3) [false discovery rate (FDR) < 0.1, and fold change > 2]. We used the Pathway Interaction Database (PID) (15) to identify key molecular pathways up-regulated in CTCs versus primary tumors, as well as those up-regulated in metastatic versus primary prostate tumors, on the basis of analyses of previously published data sets (11) (Fig. 2C, fig. S5, and table S4). In total, 21 pathways were specifically enriched in prostate CTCs, with the majority implicated in growth factor, cell adhesion, and hormone signaling (Fig. 2D and fig. S5).

Fig. 2 Gene signatures and signaling pathways in prostate CTCs.

(A) High-resolution heat map showing expression of selected gene panels in single prostate CTCs, primary tumor samples, and prostate cancer cell lines. GS, Gleason score; VCaP, a PSA-producing cell line; LNCaP.R, LNCaP cells treated with R1881; LNCaP.D, LNCaP cells treated with dimethyl sulfoxide as a vehicle control. (B) Genes differentially expressed by prostate CTCs and primary prostate tumors (FDR < 0.1, and fold change > 2). (C) PID molecular pathways (15) enriched in CTCs compared with primary tumors and in metastases compared with primary tumors (based on analysis of multiple data sets; see fig. S5 and table S4). (D) Signaling pathways enriched in prostate CTCs. Molecular pathways from the PID up-regulated in CTCs versus primary tumors (excluding those enriched in metastases compared with primary tumors), organized by PID categorization (15) (fig. S5). Abbreviations (other than proteins, clockwise from top): HDAC, histone deacetylase; AJ, adherens junction; IL2 and IL3, interleukins; ERBB1, epidermal growth factor receptor B1; TGFBR, transforming growth factor-β receptor.

The AR pathway constitutes the primary therapeutic target in prostate cancer, with specific mutations in AR (1, 16) and AR mRNA splice variants (17, 18) implicated in acquired resistance. The AR transcript was expressed (>10 rpm) in 60 out of 77 (78%) CTCs (12 out of 13 patients with prostate cancer). The T877A mutation (Thr877 replaced by Ala) in AR, previously associated with ligand promiscuity and resistance to antiandrogens (1), was identified in five out of nine CTCs from a single (1 out of 13) patient with metastatic CRPC (Fig. 3A and table S5). The F876L mutation (Phe876 replaced by Leu) in the ligand-binding domain, which converts the AR antagonist enzalutamide to a potential AR agonist (19, 20), was not detected in any of the CTCs (<1 out of 32 CTCs with sufficient sequencing reads for mutational analysis). Thus, in our study, point mutations in AR known to be associated with altered signaling were uncommon in patients with CRPC, consistent with other reports (4, 21).

Fig. 3 Heterogeneity of treatment resistance mechanisms in prostate CTCs.

(A) Heat map depicting androgen receptor (AR) abnormalities, selected signaling pathway signatures, and genes in radical prostatectomy specimens, prostate CTCs from enzalutamide-naïve patients (group A), and prostate CTCs from patients who had radiographic or biochemical progression of disease while receiving treatment with enzalutamide (group B). Noncanonical Wnt signature is from reference (15), glucocorticoid receptor (GR) signature is from reference (29), and AR signature is from reference (32) (table S7). Numbers at top of heat map represent ID numbers (Pr numbers) for patients from which each CTC is derived. (B) (Top) GSEA plots showing enrichment of noncanonical Wnt (nc-Wnt) pathway in CTCs from group B (patients with cancer progression on enzalutamide) compared with group A (enzalutamide-naïve patients). (Bottom) Enrichment of noncanonical Wnt pathway in CTCs from group B compared with group A, stratified by GR gene expression. (C) (Left) Representative micrograph (40×) of RNA-in situ hybridization assay in metastatic prostate tumors, probing for WNT5A and KRT8/18, scale bar, 50 μm. (Inset) High magnification, arrow points to WNT5A signal (red dot), arrowhead points to KRT8/18 signal (blue dot), scale bar, 10 μm. Adjacent tissue sections were probed for WNT7B, and quantification of RNA-ISH data are displayed in the table. Of nine primary tumors examined, five had >1% WNT5A expression in KRT+ cells (range 0.3%-42%) and seven had >1% WNT7B expression (range 0.5%-33.6%). Of 24 metastatic tumors examined, 16 had >1% WNT5A expression (range 0 to 50.5%) and 15 had >1% WNT7B expression (range 0 to 26%). (Right) Representative fluorescence micrographs of RNA in situ hybridization in prostate CTCs, probing for WNT5A/7B (yellow dots), and prostate CTC-specific markers (EPCAM, KLK3, FOLH1, KRT8/18/19) (red dots). DNA is stained with 4′,6′-diamidino-2-phenylindole (blue). Scale bar, 10 μm.

We then analyzed AR mRNA splice variants lacking a ligand-binding domain and encoding constitutively active proteins (1, 17). These alternative transcripts are not attributable to discrete genetic mutations, but they are commonly expressed in CRPC (4), and detection in bulk CTC preparations of the single splice variant AR-V7 has been correlated to clinical resistance to antiandrogens (18). Our single-cell analysis revealed far more complex and heterogeneous patterns of AR splice-variant expression among individual CTCs from patients with CRPC: 33 out of 73 (43%) expressed at least one type of AR splice variant (8 out of 11 CRPC patients). Among these CTCs, 26 out of 73 (36%) expressed AR-V7 (8 out of 11 patients); 18 out of 73 (25%) had a distinct splice form ARv567es (AR-V12) (8 out of 11 patients); and 7 out of 73 (10%) had AR-V1, AR-V3, or AR-V4 splice variants (5 out of 11 patients), all of which are known to result in altered signaling (Fig. 3A and table S6). Simultaneous expression of more than one type of AR splice variant was observed in 13 out of 73 (18%) single CTCs (7 out of 11 patients). In total, 7 out of 11 (64%) CRPC patients had CTCs with more than one type of AR alteration (including AR splice variants and point mutations). In contrast, no such alterations were evident in 12 primary prostate tumors, and only one out of four CTCs from two patients with castration-sensitive prostate cancer (CSPC) that was previously untreated had low-level expression of the AR-V7 splice variant (Fig. 3A and table S6). Aberrant alternative splicing is a recognized feature of many cancers (22), and indeed, another prostate-specific transcript, KLK3 (PSA) (23), showed many more alternative splice variants in CTCs from metastatic patients compared with primary tumors (P = 0.0088) (fig. S4B). Taken together, our observations indicate that intrapatient tumor heterogeneity is such that individual CTCs may have different or multiple mRNA splicing alterations.

Tumor heterogeneity is thought to increase further as second-line therapies exert additional selective pressure. We performed retrospective differential analyses in subsets of CTCs to identify mechanisms of resistance to enzalutamide, a potent AR inhibitor recently approved by the U.S. Food and Drug Administration for CRPC (24). From eight patients with metastatic prostate cancer who had not received enzalutamide (group A), 41 CTCs were compared with 36 CTCs from five patients whose cancer exhibited radiographic and/or prostate-specific antigen (PSA) progression during therapy (group B) (Fig. 3A and table S1). Gene set enrichment analysis (GSEA) of candidate PID cellular signaling pathways showed significant enrichment for noncanonical Wnt signaling in group B compared with group A CTCs (Fig. 3B and fig. S6A) (P = 0.0064; FDR = 0.239). This signaling pathway, activated by a subset of Wnt ligands, mediates multiple downstream regulators of cell survival, proliferation, and motility (fig. S6B) (2528). A separate analysis using a metagene for the PID noncanonical Wnt signature (11) (table S7) confirmed enrichment of the signature in group B compared with group A CTCs, at the level of both individual CTCs and individual patients (Fig. 3A) [P = 0.0041 (CTCs); P = 0.04 (patients)]. Among the downstream components of noncanonical Wnt, the most significantly enriched were RAC1, RHOA, and CDC42, signaling molecules involved in actin cytoskeleton remodeling and cell migration (Fig. 3A and fig. S6B) [P = 1 × 10–6 (RAC1), P = 0.0046 (RHOA), P = 0.0097 (CDC42)]. In contrast, AR abnormalities were not significantly increased among either individual CTCs or patients, when comparing enzalutamide-resistant versus enzalutamide-naïve cases, using a similar analysis (Fig. 3A).

Although most studies of CRPC have focused on acquired AR gene abnormalities, an alternative pathway, glucocorticoid receptor (GR) signaling, has recently been shown to contribute to antiandrogen resistance in a prostate cancer mouse xenograft model (29). Within our human prostate CTC data set, GR transcripts and a metagene signature of GR signaling (11) (table S7) did not reach statistical significance between patients in group A versus B [P = 0.35 (CTCs); P = 0.59 (patients)] (Fig. 3A), but an inverse relationship between GR expression and noncanonical Wnt signaling was evident. Among CTCs with low GR expression, GSEA analysis showed significant enrichment for noncanonical Wnt signaling in enzalutamide-progressing patients (group B) (P = 0.025), which was absent in CTCs with high GR expression (P = 0.34) (Fig. 3B and fig. S6D). Thus, these two AR-independent drug resistance pathways may predominate in different subsets of cancer cells.

Wnt proteins may be secreted by tumor cells as part of an autocrine loop, or they may be produced by surrounding stromal cells. We used RNA in situ hybridization (RNA-ISH) to identify the source of WNT production in tumor specimens and CTCs. Within primary untreated prostate cancers (n = 9), the noncanonical WNT5A and WNT7B mRNAs were present in a subset of tumor cells (8.9 and 11.6%, respectively), but both were rare in surrounding stromal cells (<0.2 and 0.5%, respectively) (Fig. 3C and fig. S6C). Metastatic tumor biopsies from patients with CRPC (n = 24) also had readily detectable WNT5A and WNT7B (8.0 and 6.1%, respectively) (Fig. 3C). Similarly, WNT5A or WNT7B mRNA was detected by RNA-ISH in a subset of CTCs from patients (n = 5) with CRPC (6 out of 180 CTCs; 3.3%) (Fig. 3C). Thus, a subset of prostate cancer cells express noncanonical Wnt ligands, which may provide survival signals in the context of AR inhibition.

To test whether activation of noncanonical Wnt signaling modulates enzalutamide sensitivity, we ectopically expressed the noncanonical ligands WNT4, WNT5A, WNT7B, or WNT11 in LNCaP androgen-sensitive human prostate cancer cells, which express low endogenous levels (fig. S7, A and B). Survival of the AR-positive LNCaP cells in the presence of enzalutamide was enhanced by the noncanonical Wnt ligands, particularly WNT5A (Fig. 4A) (P = 2.8 × 10−5) (fig. S7C). Remarkably, endogenous WNT5A was acutely induced upon treatment with enzalutamide, suggestive of a feedback mechanism, and its depletion (knockdown) resulted in reduced cell proliferation (Fig. 4B and fig. S7D) (P = 6.6 × 10−4). We also generated stable enzalutamide-resistant LNCaP cells through prolonged in vitro selection (fig. S7E). These cells also exhibited increased expression of endogenous WNT5A, whose suppression reduced proliferation in enzalutamide-supplemented medium (Fig. 4C) (P = 0.005) (fig. S7F). Finally, we tested the contribution of noncanonical Wnt to antiandrogen resistance in an independent data set, interrogating the previously published mouse LNCaP xenograft model, in which aberrant activation of GR contributes to enzalutamide resistance (29). A significant association between enzalutamide resistance and noncanonical Wnt signaling was evident (P = 0.023), which again showed an inverse relation between GR expression and noncanonical Wnt signaling (P = 0.032 for GR low versus P = 0.11 for GR high) (Fig. 4D and fig. S8, A and B). This independent data set further validates the independent contributions of GR and noncanonical Wnt signaling to antiandrogen resistance.

Fig. 4 Noncanonical Wnt signaling and enzalutamide resistance.

(A) Ectopic expression of noncanonical Wnt in LNCaP cells increases cell survival in the presence of enzalutamide (3 μM). (B) Enzalutamide treatment induces WNT5A mRNA expression in LNCaP cells, and WNT5A suppression in enzalutamide-treated LNCaP cells results in decreased proliferation. (C) Stable enzalutamide-resistant cells (LN_EnzR), derived from LNCaP cells, express increased levels of WNT5A, and WNT5A small interfering RNA results in reduced proliferation in the presence of enzalutamide. Each experiment (A) to (C) was performed at least three times. Data are presented as means ± SD. (D) (Left) GSEA plot showing enrichment of noncanonical Wnt pathway in mouse xenografts derived from enzalutamide-resistant LREX′ cells (29) compared with control xenografts derived from LNCaP/AR cells (data from LREX′ and Con B entries, GEO GSE52169). (Right) GSEA plots showing noncanonical Wnt pathway enrichment in antiandrogen-resistant xenografts, when stratified by GR gene expression (data from Res and Con A entries, GEO GSE52169).

In summary, by RNA profiling single prostate CTCs, we demonstrate their differences from primary tumors, as well as their heterogeneity within individual patients. The acquisition of AR-dependent and AR-independent alterations conferring resistance to antiandrogen therapies is also heterogeneous. Among AR alterations, more than half of all patients had multiple AR splice variants present within different CTCs and about 1 out of 6 of single cancer cells had simultaneous expression of several AR splice variants. Two AR-independent pathways, activation of GR and noncanonical Wnt signaling, coexist in different subsets of cells. Wnt signaling has been implicated in multiple cellular functions linked to prostate cancer progression (4, 2528), and noncanonical Wnt signaling may be targeted by suppression of its key downstream components, such as Rho kinase (30). Our study is limited by its retrospective nature and relatively small sample size (13 patients; average of six CTCs per patient), a consequence of the rarity of intact CTCs and inefficiencies inherent in manual single-cell micromanipulation techniques, obstacles that might be overcome with future improvements in CTC isolation and single-cell sequencing technologies. Nevertheless, the heterogeneity of CTCs in patients with CRPC stands in contrast to the striking homogeneity of AR signaling in single CTCs from untreated patients (5). Although these observations require validation in prospective trials, they point to complex and heterogeneous drug resistance mechanisms in advanced prostate cancer, which may affect therapeutic efficacy.

Supplementary Materials

Materials and Methods

Figs. S1 to S8

Tables S1 to S7

References (3337)

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank C. Sawyers for helpful discussions; A. McGovern, E. Stadtmueller, and B. Abebe for clinical trial support; and L. Libby and L. Nieman for technical assistance. This work was supported by grants from the Prostate Cancer Foundation (D.A.H., S.M., M.T., M.R.S., and R.J.L.), Charles Evans Foundation (D.A.H.), Department of Defense (D.T.M., R.J.L., and D.T.T.), Stand Up to Cancer (D.A.H., M.T., S.M., and L.V.S.), Howard Hughes Medical Institute (D.A.H.), National Institute of Biomedical Imaging and Bioengineering (NIBIB), NIH, EB008047 (M.T.), NCI 2RO1CA129933 (D.A.H.), National Cancer Institute, NCI, Federal Share Program and Income (S.M. and D.T.M.), Affymetrix, Inc. (D.T.T., K.A., and N.D.), Mazzone Program–Dana-Farber Harvard Cancer Center (D.T.M.), Burroughs Wellcome Fund (D.T.T.), and the Massachusetts General Hospital–Johnson & Johnson Center for Excellence in CTC Technologies (D.A.H., M.T., and S.M.). D.T.T. is a paid consultant for Affymetrix, Inc.; R.J.L. is a paid consultant for Janssen LLC. The Massachusetts General Hospital has filed for patent protection for the CTC-iChip technology. RNA-sequencing data have been deposited in GEO under accession number GSE67980.
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