Regulation of stem/progenitor cell maintenance by BMP5 in prostate homeostasis and cancer initiation.

Tissue homeostasis relies on the fine regulation between stem and progenitor cell maintenance and lineage commitment. In the adult prostate, stem cells have been identified in both basal and luminal cell compartments. However, basal stem/progenitor cell homeostasis is still poorly understood. We show that basal stem/progenitor cell maintenance is regulated by a balance between BMP5 self-renewal signal and GATA3 dampening activity. Deleting Gata3 enhances adult prostate stem/progenitor cells self-renewal capacity in both organoid and allograft assays. This phenotype results from a local increase in BMP5 activity in basal cells as shown by the impaired self-renewal capacity of Bmp5-deficient stem/progenitor cells. Strikingly, Bmp5 gene inactivation or BMP signaling inhibition with a small molecule inhibitor are also sufficient to delay prostate and skin cancer initiation of Pten-deficient mice. Together, these results establish BMP5 as a key regulator of basal prostate stem cell homeostasis and identifies a potential therapeutic approach against Pten-deficient cancers.

Introduction

Maintaining homeostasis in adult tissues requires a fine balance between stem cell maintenance and differentiation (Morrison and Kimble, 2006). An amplification of the stem/progenitor cell pool at the expense of cell differentiation or, conversely, the depletion of the stem/progenitor cell pool by premature differentiation are both expected to be detrimental to tissue homeostasis (Signer and Morrison, 2013; Tomasetti and Vogelstein, 2015). The dynamic process of stem/progenitor cell maintenance is regulated by signals from the microenvironment (niche) and by intrinsic regulatory mechanisms (Dumont et al., 2015). Similarly, cancer initiation and progression are thought to rely in large part on the self-renewal potential of cancer stem cells (Batlle and Clevers, 2017). In many systems, including the prostate, the molecular and cellular mechanisms regulating stem/progenitor cell homeostasis are still poorly understood.

The prostate consists of a multitude of branched epithelial ducts funneling prostatic fluids to the upper urethra. Prostatic ducts are composed of an inner layer of secretory luminal cells surrounded by a layer of basal cells interspersed with rare neuroendocrine cells. Tissue homeostasis and regeneration of the prostate have been shown to rely on endogenous stem cell populations. Allograft assays and lineage-tracing experiments revealed the presence of stem cells in both the basal and luminal compartments (Choi et al., 2012; Goldstein et al., 2010; Wang et al., 2013). During homeostasis and regeneration, adult stem cells are largely unipotent (Choi et al., 2012; Liu et al., 2011; Wang et al., 2013). However, basal stem cells act as facultative stem cells by activating basal to luminal lineage conversion under various stress conditions (Kwon et al., 2014; Toivanen et al., 2016). In contrast, basal stem cells are constitutively multipotent in the developing prostate, where they populate the luminal cell layer through asymmetric cell divisions (Ousset et al., 2012; Shafer et al., 2017; Wang et al., 2014a). In prostate cancer, basal and luminal stem cells can both act as tumor-initiating cells and are thought to contribute to tumor recurrence (Choi et al., 2012; Goldstein et al., 2010; Wang et al., 2009; Wang et al., 2013; Zhao et al., 2018).

In recent years, GATA transcription factors have emerged as important regulators of prostate development and cancer (Nguyen et al., 2013; Shafer et al., 2017; Xiao et al., 2016). During postnatal development, GATA3 is expressed in both basal and luminal cells and plays an important role in lineage specification and tissue organization (Shafer et al., 2017). In cancer, nuclear loss of GATA3 is associated with the progression to castration-resistant prostate cancer (CRPC) and poor prognosis (Nguyen et al., 2013). Accordingly, Pten-deficient mouse model of prostate cancer exhibits a progressive loss of Gata3 expression (Nguyen et al., 2013). In this model, prostate cancer could be accelerated by an acute loss of Gata3, or significantly delayed by GATA3 maintenance through transgenic expression (Nguyen et al., 2013). Despite this growing body of evidence of the importance of GATA3 in the prostate, its role in adult prostate stem/progenitor cell homeostasis is currently unknown.

Among the established regulators of stem cell homeostasis is the BMP signaling pathway (Oshimori and Fuchs, 2012). BMPs are part of the TGFβ family of signaling molecules, a group comprised of key regulators of cell differentiation, apoptosis, epithelial-mesenchymal transition and stem cell homeostasis (David and Massagué, 2018). BMPs are typically expressed in the stem cell niche and promote either stem cell quiescence or self-renewal depending on context (Genander et al., 2014; Haramis et al., 2004; He et al., 2004; Tadokoro et al., 2016; Tian and Jiang, 2014). In prostate cancer, BMP6 signaling appears to promote cancer progression (Darby et al., 2008; Lu et al., 2017), while BMP7 induces quiescence in metastatic cancer cells (Kobayashi et al., 2011). Yet, possible regulation of adult prostate stem cells by BMPs has received little attention so far.

Here, we explore the mechanisms of adult prostate stem/progenitor cell homeostasis. Combining mouse genetics, organoid cultures and RNA-seq analysis, we identify a crucial role for GATA3 in the control of basal stem/progenitor cell maintenance and identify BMP5 as a key effector of this activity. We further demonstrate that targeting the BMP pathway with a small molecule inhibitor significantly slows down cancer progression in the prostate as well as in the skin.

Results

Gata3 deficiency increases the long-term maintenance of prostate stem/progenitor cells

Gata3 has previously been reported to play a role in prostate development (Shafer et al., 2017) and in cancer progression (Nguyen et al., 2013). Whether Gata3 also regulates adult prostate stem cell homeostasis remains unknown. To explore this possibility, we first examined the expression pattern of Gata3 in adult prostate lineages using the surface markers Lin(CD31,TER119,CD45); SCA1; CD49f; EpCAM; TROP2-, to separate basal, luminal and stromal cells (Figure 1—figure supplement 1A,B and C). Taking advantage of a Gata3 knock-in reporter mouse strain, we found Gata3-driven GFP expression in both the basal and luminal populations (Figure 1—figure supplement 1B). This finding was confirmed by qRT-PCR analysis (Figure 1—figure supplement 1C) and by immunofluorescence staining of GATA3 in basal and luminal cells (Figure 1—figure supplement 1D).

Figure 1—figure supplement 1.

GATA3 is expressed in prostate basal and luminal cells.

(A) Representative fluorescence-activated cell sorting (FACS) strategy to purify stromal, luminal and basal cell populations from adult prostate using antibodies against surface markers: CD45, CD31, TER119, CD49f, EpCAM, SCA1, and TROP2. (B) Histogram of endogenous Gata3-driven GFP reporter expression in the indicated populations in Gata3 knock-in and wild-type mice. (C) Expression of specific population markers in FACS sorted stromal, luminal and basal cell populations from adult prostate as assessed by quantitative RT-PCR. Relative mRNA expression levels are normalized to Ppia mRNA levels (Average ± SD, n = 3). (D) Immunofluorescence analysis of GATA3 in prostate tissue from adult wild-type mice. Notice the expression of GATA3 in both basal and luminal cells. Scale bar: 50 μm. (E) Specific activation of Rosa26LstopLTdTomato fluorescent reporter by Pbsn-Cre transgene in basal and luminal populations from adult prostate tissue as assessed by FACS.

We next sought to determine whether GATA3 plays a role in prostate stem/progenitor cell homeostasis. For this, we purified basal cells from wild type, Pbsn-Cre Gata3 or Pbsn-Cre Rosa26 adult prostates and performed a short-term organoid propagation assay where cultures were passaged after 7 days in order to specifically look at their propagation potential. Pbsn-Cre mice express the Cre recombinase in both basal and luminal cells of the prostate (Figure 1—figure supplement 1E; Wu et al., 2011). In this assay, short-term organoids grown from wild type basal cells could be passaged for three to five generations, as the organoids progressively lose their propagation potential (Figure 1A–B). Interestingly, cells obtained from Pbsn-Cre Rosa26 mice, which express higher levels of GATA3 upon Cre-mediated deletion of a stop cassette (Nguyen et al., 2013), had a reduced organoid propagation potential (Figure 1A and Figure 1—figure supplement 2A). In striking contrast, cells derived from Gata3-deficient prostates (Pbsn-Cre Gata3) (Grote et al., 2006) showed an increased organoid-forming potential over several passages (Figure 1A and Figure 1—figure supplement 2A-B). Organoid size, proliferation and apoptosis levels were unaffected by Gata3 expression levels (Figure 1—figure supplement 2C–D-E), indicating that the stem/progenitor maintenance potential rather than cell proliferation or survival is altered in those organoids. To study the effect of Gata3 loss on cell differentiation, testosterone was added to the media to favor differentiation of Pbsn-Cre Gata3 prostate organoids which revealed a decrease in organoids capable of forming lumens, pointing to a cell differentiation defect associated with the increase in organoid-forming potential (Figure 1—figure supplement 2F; Figure 1—figure supplement 2G).

Figure 1.

Gata3 loss leads to an expansion of prostate basal stem/progenitor cells numbers.

(A) Effect of Gata3 loss and overexpression on in vitro basal stem/progenitor maintenance potential. Organoid-forming potential was assessed by plating equal numbers (105 cells) of sorted basal prostate cells from the indicated genotypes in Matrigel and passaged every 7 days. Shown is the absolute number of cells obtained after each passage for the indicated genotypes. Data are representative of two independent experiments from a pool of prostate cells from a minimum of three mice. (B) Specific deletion of Gata3 in KRT5+ basal cells affect the organoid-forming potential upon passage. Organoid-forming potential was assessed as in (A). Cre activity was induced in vitro by treatment with hydroxy-tamoxifen for the first passage. (C) Gata3 loss increase regenerative capacity in vivo. Different numbers of sorted basal cells from wild type (Pbsn-Cre, Pbsn-Cre Gata3 and Krt5) prostate were mixed with UGSM and transplanted under the kidney capsule of immunodeficient mice. All mice contain Rosa26/+ allele and Cre activity was induced in vivo by tamoxifen injection in adult mice 4 weeks prior to organoid propagation potential assessment. Prostate reconstituting units (PRU) frequency of total basal cells was calculated based on growth of TdTomato+ grafts using the Limiting Dilution Analysis software L-Calc (StemCell Technologies) according to Poisson statistics (two-tailed t-test; *p<0.05, **p<0.001). (D) Immunofluorescence staining of lineage-specific markers KRT5 (basal) and KRT8/18 (luminal) in wild type, Pbsn-Cre Gata3 and Krt5 allografts. Scale bar is representative of 50 μm. See also Figure 1—figure supplements 1–2.

(A) Representative fluorescence-activated cell sorting (FACS) strategy to purify stromal, luminal and basal cell populations from adult prostate using antibodies against surface markers: CD45, CD31, TER119, CD49f, EpCAM, SCA1, and TROP2. (B) Histogram of endogenous Gata3-driven GFP reporter expression in the indicated populations in Gata3 knock-in and wild-type mice. (C) Expression of specific population markers in FACS sorted stromal, luminal and basal cell populations from adult prostate as assessed by quantitative RT-PCR. Relative mRNA expression levels are normalized to Ppia mRNA levels (Average ± SD, n = 3). (D) Immunofluorescence analysis of GATA3 in prostate tissue from adult wild-type mice. Notice the expression of GATA3 in both basal and luminal cells. Scale bar: 50 μm. (E) Specific activation of Rosa26LstopLTdTomato fluorescent reporter by Pbsn-Cre transgene in basal and luminal populations from adult prostate tissue as assessed by FACS.

(A–B) Growth rate of cells upon organoid passage for the indicated genotypes calculated from nonlinear regression curve fit of data from Figure 1A and B, respectively (one-way ANOVA; *p<0.02, **p<0.002, ***p<0.0001). (C) Loss of Gata3 does not affect organoid size over several passages. Shown is the average diameter of wild type and Pbsn-Cre Gata3 organoids (Average ± SD). (D–E) Loss of Gata3 in organoids does not affect proliferation or survival. Immunofluorescence staining for KRT5 and Ki67 (D) and TUNEL reaction (E) was done on 4-day-old organoids at passage 2. (F–G) Gata3 loss reduces cell differentiation potential. Organoids from wild type and Pbsn-Cre Gata3 grown for 7 days were passaged and differentiated by dihydrotestosterone (DHT) treatment. The average percentage of organoids which form lumen (F) and representative images of organoids with and without lumen (G) for the indicated genotypes (average ± SD, n = 117, two-tailed t-test as compared to wild-type condition; *p<0.0001).

(A–B) KRT5 (basal cells) and KRT8/18 (luminal cell) expression in day 7 (A) and day 14 (B) wild-type prostate organoids. Shown is the H&E staining of a day 14 organoid. (C) TdTomato+ allograft. Purified Lin-SCA1+CD49f+EpCAM+TROP2+ basal prostate cells from Pbsn-Cre Rosa26 mice were mixed with urogenital sinus mesenchyme cells (UGSM) and transplanted under the kidney capsule of immunodeficient mice. Shown is the brightfield (left) as well as the fluorescent (right) picture of the allograft tissue after 90 days. (D) Immunofluorescence analysis of lineage specific markers KRT5 and KRT8/18 in wild type, Pbsn-Cre Gata3 and Krt5 allografts. Lower magnification pictures of allograft from Figure 1D.

Figure 1—figure supplement 2.

Gata3 is important for propagation and differentiation of organoids.

(A–B) Growth rate of cells upon organoid passage for the indicated genotypes calculated from nonlinear regression curve fit of data from Figure 1A and B, respectively (one-way ANOVA; *p<0.02, **p<0.002, ***p<0.0001). (C) Loss of Gata3 does not affect organoid size over several passages. Shown is the average diameter of wild type and Pbsn-Cre Gata3 organoids (Average ± SD). (D–E) Loss of Gata3 in organoids does not affect proliferation or survival. Immunofluorescence staining for KRT5 and Ki67 (D) and TUNEL reaction (E) was done on 4-day-old organoids at passage 2. (F–G) Gata3 loss reduces cell differentiation potential. Organoids from wild type and Pbsn-Cre Gata3 grown for 7 days were passaged and differentiated by dihydrotestosterone (DHT) treatment. The average percentage of organoids which form lumen (F) and representative images of organoids with and without lumen (G) for the indicated genotypes (average ± SD, n = 117, two-tailed t-test as compared to wild-type condition; *p<0.0001).

To confirm that the increased organoid-forming potential of Gata3-deficient cells is intrinsic to basal cells, we used the tamoxifen-inducible Krt5 knock-in allele. In vitro activation of CreERT2 in KRT5+ cells reproduced the results obtained with the Pbsn-Cre strain (Figure 1B and Figure 1—figure supplement 2B). We further assessed the possibility that the increased organoid propagation potential comes from luminal cells generated during organoid growth and differentiation (Figure 1—figure supplement 3A–B). For this, we specifically inactivated Gata3 in vitro in luminal cells using Krt8 transgene. This did not lead to an increase in organoid-forming potential (Figure 1B and Figure 1—figure supplement 2B), indicating that the role of GATA3 in basal stem/progenitor cell homeostasis is restricted to the basal compartment.

Figure 1—figure supplement 3.

Ductal structures with multiple alveoli and a lumenized epithelial structure in allografts and organoids.

(A–B) KRT5 (basal cells) and KRT8/18 (luminal cell) expression in day 7 (A) and day 14 (B) wild-type prostate organoids. Shown is the H&E staining of a day 14 organoid. (C) TdTomato+ allograft. Purified Lin-SCA1+CD49f+EpCAM+TROP2+ basal prostate cells from Pbsn-Cre Rosa26 mice were mixed with urogenital sinus mesenchyme cells (UGSM) and transplanted under the kidney capsule of immunodeficient mice. Shown is the brightfield (left) as well as the fluorescent (right) picture of the allograft tissue after 90 days. (D) Immunofluorescence analysis of lineage specific markers KRT5 and KRT8/18 in wild type, Pbsn-Cre Gata3 and Krt5 allografts. Lower magnification pictures of allograft from Figure 1D.

We next tested the capacity of GATA3 to regulate stem/progenitor cell homeostasis in vivo by allograft assay. To this end, basal cells from wild type, Pbsn-Cre Gata3 and Krt5 prostates (all expressing Rosa26 to trace donor tissues) were implanted with urogenital sinus mesenchyme (UGSM) (Goldstein et al., 2010; Wang et al., 2009) under the renal capsule of host mice and grown for 90 days in the presence of exogenous testosterone. Using serial dilution, we assessed the proportion of basal cells capable of generating TdTomato+ prostate tissue [measured as prostate reconstituting units (PRU)] (Figure 1C and Figure 1—figure supplement 3C). The PRU frequency in the basal population was found to be 1 per 8600 for control. In contrast, Pbsn-Cre Gata3 gave an average of 1 in 3,942, and Krt5 of 1 in 1879 which corresponds respectively to over two- and four-fold increase in steady state stem/progenitor cell numbers in the Gata3-deficient basal cell pool. The difference between Pbsn-Cre and Krt5CreERT2 likely reflects the deletion efficiency of both transgenic lines in basal cells. The histological examination of these grafts showed ductal structures with multiple alveoli and a lumenized epithelial structure composed of a bilayer of basal and luminal cells as evidenced by KRT5 and KRT8/18 staining (Figure 1D and Figure 1—figure supplement 3D).

From these results, we conclude that GATA3 is critical for regulating basal stem/progenitor cell maintenance in the prostate. GATA3 inactivation promotes an increase in self-renewing capacity, leading to a gradual expansion of the stem/progenitor cell pool.

BMP signaling is upregulated in Gata3-deficient organoids

To gain insight into the molecular mechanisms leading to the amplification of the stem cell pool upon Gata3 inactivation, we performed RNA-seq on wild type and Pbsn-Cre Gata3 prostate organoids harvested after 0, 2, 3 and 4 passages (Figure 2A). To track the deletion of Gata3 exon four by the Pbsn-Cre transgene, we mapped RNA-seq transcript to the Gata3 locus (Figure 2B). Surprisingly, cells from passage 0 mostly retained the floxed exon 4 of Gata3. However, loss of exon four occurred in about 50% of cells at passage 2, and in the vast majority of cells from passages 3 and 4. This finding, likely due to a limited efficiency of Pbsn-Cre in basal cells, allowed us to use the dynamics of locus deletion as an additional filter to identify GATA3 regulated genes.

Figure 2.

Bmp5 expression in organoids is regulated by Gata3.

(A) Schematic of RNA-seq strategy. mRNA was isolated from 4 days old wild type and Pbsn-Cre Gata3 organoids at passages P0, P2, P3 and P4. (B) Deletion of exon four in Pbsn-Cre Gata3 samples increases with passages. Shown are the read counts from RNAseq assigned to the Gata3 locus in samples isolated from wild type and Pbsn-Cre Gata3 prostate tissue at passage P0, P2, P3 or P4. (C) Venn diagram of genes differentially expressed between wild type and Pbsn-Cre Gata3 prostate organoids using likelihood-ratio test with q-value <0.01. (D) Heatmap of log2 transformed mRNA read counts of differentially expressed genes between wild type and Pbsn-Cre Gata3 organoids and whose expression pattern follows Gata3 loss with passages. (E) Bmp5 mRNA expression levels as assessed by quantitative RT-PCR in both wild type and Pbsn-Cre Gata3 organoids over passages. Data represent the average ± SD from three independent cDNA obtained from a pool of prostate cells from a minimum of three mice. Relative mRNA expression levels are normalized to Ppia mRNA levels (two-tailed t-test as compared to wild-type condition; *p<0.01, **p<0.005). See also Figure 2—figure supplements 1–2.

(A) Heatmap of log2 transformed mRNA read counts of genes differentially expressed between wild type and Pbsn-Cre Gata3 organoids at passages 3 and 4 using likelihood-ratio test (q-value <0.01). (B) Overexpression of GATA3 in organoids reduces Bmp5 expression. Shown is the relative expression levels of Bmp5 as assessed by RNAseq from 4-day-old organoids of the indicated passages and genotypes. Data are presented as fold expression over wild-type levels at passage 0. (C) Gata3-dependent gene signature is enriched (A) in genes associated with BMP signaling from different libraries using Enrichr software: targets computed from ENCODE and ChEA project ChIP-seq datasets (ENCODE and ChEA consensus), protein-protein interactions network (PPIs), genes coexpressed with transcription factors (ARCHS4) and genes affected by gene mutation from Gene Expression Omnibus datasets (GEO).

(A) The expression of Bmp5 in sorted basal, luminal and stromal cells from wild type and Pbsn-Cre Gata3 prostates. Relative expression levels normalized to Ppia and wild-type basal cells as assessed by quantitative RT-PCR (Average ± SD, n = 3), (two-tailed t-test as compared to wild-type condition; *p<0.0001). (B) ChIP-seq data from the Gene transcription regulation database (GTRD) showing regions of the Bmp5 (mouse, upper pannel) and BMP5 (human, lower pannel) locus bound by GATA3 protein. (C) Bmp5 locus is bound by GATA3 protein in prostate cells. Crosslinked extract from Gata3 prostate tissue were subjected to BioChIP pulldown and amplification of the indicated locus was done by quantitative PCR. Shown is the fold enrichment of streptavidin bead over Igg control beads (average ± SD, two-tailed t-test as compared to Cdh1 promoter; *p<0.001, **p<0.0001).

Figure 2—figure supplement 1.

Gata3-dependent gene signature.

(A) Heatmap of log2 transformed mRNA read counts of genes differentially expressed between wild type and Pbsn-Cre Gata3 organoids at passages 3 and 4 using likelihood-ratio test (q-value <0.01). (B) Overexpression of GATA3 in organoids reduces Bmp5 expression. Shown is the relative expression levels of Bmp5 as assessed by RNAseq from 4-day-old organoids of the indicated passages and genotypes. Data are presented as fold expression over wild-type levels at passage 0. (C) Gata3-dependent gene signature is enriched (A) in genes associated with BMP signaling from different libraries using Enrichr software: targets computed from ENCODE and ChEA project ChIP-seq datasets (ENCODE and ChEA consensus), protein-protein interactions network (PPIs), genes coexpressed with transcription factors (ARCHS4) and genes affected by gene mutation from Gene Expression Omnibus datasets (GEO).

Figure 2—figure supplement 2.

Bmp5 expression is specifically affected in Gata3-deficient basal cells.

(A) The expression of Bmp5 in sorted basal, luminal and stromal cells from wild type and Pbsn-Cre Gata3 prostates. Relative expression levels normalized to Ppia and wild-type basal cells as assessed by quantitative RT-PCR (Average ± SD, n = 3), (two-tailed t-test as compared to wild-type condition; *p<0.0001). (B) ChIP-seq data from the Gene transcription regulation database (GTRD) showing regions of the Bmp5 (mouse, upper pannel) and BMP5 (human, lower pannel) locus bound by GATA3 protein. (C) Bmp5 locus is bound by GATA3 protein in prostate cells. Crosslinked extract from Gata3 prostate tissue were subjected to BioChIP pulldown and amplification of the indicated locus was done by quantitative PCR. Shown is the fold enrichment of streptavidin bead over Igg control beads (average ± SD, two-tailed t-test as compared to Cdh1 promoter; *p<0.001, **p<0.0001).

The comparison of differentially regulated genes between wild type and Pbsn-Cre Gata3 samples at passages 3 and 4 identified 205 candidate genes (Figure 2C and Figure 2—figure supplement 1A). Of interest, Bmp5 emerged as the candidate with the highest differential expression ratio between wild type and mutant at passages 2, 3 and 4 (up to 48-fold difference), while being unaffected at passage 0, when the Gata3 locus is still intact (Figure 2D and Figure 2—figure supplement 1B). A quantification of Bmp5 levels by qRT-PCR validated the RNA-seq results (Figure 2E). In line with the dynamics of Bmp5 expression, the analysis of all differentially expressed genes with the ENRICHR resource (Chen et al., 2013; Kuleshov et al., 2016) identified a number of genes regulated by SMAD, BMP, BMPR1a- and BMPR2 (Software ARCH2 and GEO datasets; Figure 2—figure supplement 1C) as well as a strong enrichment for SMAD4 binding sites in the regulatory region of those genes (Software Encode-ChEA; Figure 2—figure supplement 1C).

On the grounds of the consistent association between the GATA3 response in prostate stem/progenitor cells and the BMP/SMAD signaling pathway, we decided to probe further the role of BMP5 in prostate stem cell regulation.

BMP signaling regulates basal stem/progenitor cell maintenance

Previous studies have linked canonical BMP signaling to progenitor cell proliferation and differentiation in the epidermis, hair follicle and intestine (Genander et al., 2014; Haramis et al., 2004; He et al., 2004; Lewis et al., 2014). However, relatively little is known about the regulation of stem cell homeostasis by BMPs, notably in the prostate. To better understand the source of BMP5 in the adult prostate, we measured Bmp5 expression by qRT-PCR in basal, luminal and stromal cells sorted from wild type and Pbsn-Cre Gata3 prostates (Figure 2—figure supplement 2A). In wild-type prostates, Bmp5 was found to be expressed in all three cell types (Figure 2—figure supplement 2A). Interestingly, Gata3 inactivation primarily increased Bmp5 expression in basal cells, which suggests a specific role for Gata3 in this compartment. We next sought to determine whether GATA3 directly binds the Bmp5 locus. For this, we first probed ChIP-seq data from the Gene transcription regulation database (GTRD), which identified several regions of the Bmp5 locus bound by GATA3 in murine and human cells (Figure 2—figure supplement 2B). To validate this possibility in prostatic tissue, we took advantage of a biotinylated allele of Gata3 (Gata3) and performed BioChIP-PCR assay on adult prostates. Using this high-affinity system, we found that GATA3 is bound to the Bmp5 locus which suggest that GATA3 regulates directly Bmp5 expression in prostate basal cells (Figure 2—figure supplement 2C). In order to clarify the genetic relationship between Gata3 and Bmp5, we used the ‘small ear’ (SE) mouse strain, which harbor a nonsense mutation in the propeptide region of Bmp5 that prevents mature protein expression (King et al., 1994; Figure 3—figure supplement 1A). We found no difference in Gata3 expression levels in basal cells in the absence of Bmp5 both by qRT-PCR and by FACS using Gata3 knock-in reporter mouse strain on a wild type or Bmp5 background (Figure 3—figure supplement 1B–C), suggesting that BMP5 is not a critical regulator of Gata3 expression in the prostate.

Figure 3—figure supplement 1.

GATA3 expression is not regulated by Bmp5 expression in the prostate.

(A) Schematic of BMP5 protein showing the Small Ear (SE) mutation which lead to the introduction of an early stop codon in the propeptide region. (B) Gata3 levels are not affected by Bmp5 loss. Relative expression levels of Gata3 was normalized as in Figure 2E. (C) Representative histogram of endogenous Gata3-driven GFP reporter expression in the basal population of whole prostate from the indicated genotype. (D) Immunofluorescence staining for KRT5 and TUNEL reaction was done on wild type and Bmp5 4-day-old organoids at passage 2. Scale bar is representative of 50 μm.

To test whether BMP signaling affects the self-renewal potential of Gata3-deficient basal cells, we blocked it with the inhibitory protein NOGGIN in culture. In wild-type organoids, NOGGIN treatment prevented basal cells from being passaged past four generations (Figure 3A and Figure 3—figure supplement 2A). Strikingly, NOGGIN treatment also abrogated the long-term amplification of Gata3-deficient organoids, suggesting that BMP signaling is a key mediator of the Gata3-deficient propagation phenotype (Figure 3A and Figure 3—figure supplement 2A). This decrease in organoid-forming potential was not caused by changes in proliferation nor apoptosis, as shown by unaltered Ki67 and TUNEL stainings in treated organoids (Figure 3—figure supplement 2E–F). To validate these result, we used K02288, a potent small molecule inhibitor selective to BMPR-SMAD1/5/8 signaling (Sanvitale et al., 2013) which showed similar results to NOGGIN treatment (Figure 3B and Figure 3—figure supplement 2B).

Figure 3.

BMP5 increases the propagation potential of basal stem/progenitor cells.

(A–B) Organoid-forming capacity is abrogated by both BMP and BMPR-SMAD1/5/8 inhibitors treatment. Sorted basal cells from wild type or Krt5 prostate were grown in presence or absence of NOGGIN (A) or small inhibitor K02288 (B) for six passages. Cre activity was induced by hydroxy-tamoxifen treatment in vitro for the first passage (A) or by tamoxifen injection in vivo 4 weeks prior to culture (B). Organoid-forming potential was assessed as in Figure 1A. (C) Bmp5 loss reduces organoid-forming activity in vitro, while propagation potential capacity of wild type cells is increased by BMP5 treatment. Basal cells (105) from wild type, Bmp5, Krt5 or Krt5 mice were grown for six passages. Exogenous BMP5 was added to culture media where indicated. Cre activity was induced in vivo as in (B). (D) Bmp5 silencing specifically affects organoid-forming activity in vitro. ShRNA against Bmp5 or scrambled were electroporated in first passage organoid from wild type and Krt5 basal cells and grown for seven passages. Cre activity was induced in vivo as in (B). (E) Bmp5 levels affects regenerative potential in vivo. Different numbers of sorted basal prostate cells from Rosa26 (control) or Bmp526LTdTomato were transplanted with UGSM either wild type, ectopically expressing BMP5 or derived from Bmp5 mice. Limiting dilution analysis was done as in Figure 1C. Notice that the loss of Bmp5 in basal cells but not in UGSM affects prostate reconstituting units (PRU) frequency (two-tailed t-test as compared to wild-type condition; **p<0.02, *p<0.04). (F) Immunofluorescence of allografts show presence of prostatic ducts expressing both KRT5 and KRT8/18. Scale bar is representative of 50 μm. See also Figure 3—figure supplements 1–2.

(A) Schematic of BMP5 protein showing the Small Ear (SE) mutation which lead to the introduction of an early stop codon in the propeptide region. (B) Gata3 levels are not affected by Bmp5 loss. Relative expression levels of Gata3 was normalized as in Figure 2E. (C) Representative histogram of endogenous Gata3-driven GFP reporter expression in the basal population of whole prostate from the indicated genotype. (D) Immunofluorescence staining for KRT5 and TUNEL reaction was done on wild type and Bmp5 4-day-old organoids at passage 2. Scale bar is representative of 50 μm.

(A–D) Growth rate of cells upon organoid passage for the indicated genotypes and treatment calculated from nonlinear regression curve fit of data from Figure 3A–D, respectively (one-way ANOVA; *p<0.02, **p<0.005, ***p<0.0005, ****p<0.0001). (E–F) Treatment of organoids with BMP5 or NOGGIN does not affect proliferation nor apoptosis. Immunofluorescence staining for KRT5 and Ki67 (E) or TUNEL reaction (F) were done on wild-type 4-day-old organoids treated or not with BMP5 or NOGGIN at passage 2. Scale bar is representative of 50 μm.

Immunofluorescence analysis of lineage-specific markers KRT5 and KRT8/18 in indicated allografts. Lower magnification pictures of allograft from Figure 3D.

Figure 3—figure supplement 2.

BMP5 treatment does not affect proliferation nor survival in organoid.

(A–D) Growth rate of cells upon organoid passage for the indicated genotypes and treatment calculated from nonlinear regression curve fit of data from Figure 3A–D, respectively (one-way ANOVA; *p<0.02, **p<0.005, ***p<0.0005, ****p<0.0001). (E–F) Treatment of organoids with BMP5 or NOGGIN does not affect proliferation nor apoptosis. Immunofluorescence staining for KRT5 and Ki67 (E) or TUNEL reaction (F) were done on wild-type 4-day-old organoids treated or not with BMP5 or NOGGIN at passage 2. Scale bar is representative of 50 μm.

To determine whether BMP5 is sufficient to promote basal stem/progenitor cell maintenance, we treated wild-type basal prostate organoids with exogenous BMP5 over several passages. As expected, BMP5 treatment increased the organoid-forming capacity of basal cells over time (Figure 3C and Figure 3—figure supplement 2C). Conversely, Bmp5 cells showed an impaired organoid-forming potential comparable to NOGGIN or K02288-treated cells (Figure 3C and Figure 3—figure supplement 2C). Here again, neither survival (Figure 3—figure supplement 1D) nor proliferation was affected in the organoids that successfully grew, which supports a self-renewal phenotype. We next assessed whether Gata3-deficient phenotype was specifically dependent on the BMP5 ligand by inactivating both Bmp5 and Gata3 in KRT5-positive basal cells in vivo. Bmp5 mutation impaired the increased organoid-forming capacity of Gata3-deficient cells (Figure 3C and Figure 3—figure supplement 2C). In addition, the acute loss of Bmp5 using shRNA against Bmp5 showed a similar effect, inhibiting the passaging capacity of both wild type and Gata3-deficient organoid (Figure 3D and Figure 3—figure supplement 2D). Together, these results confirm that the increased propagation potential of Gata3-deficient basal cells requires BMP signaling driven by the BMP5 ligand.

To validate this phenotype in vivo, we assessed whether BMP5 is sufficient to promote prostate tissue development from basal stem/progenitor cells in an allograft assay. For this, we compared the regenerative potential of wild-type basal cells (expressing the constitutively active Rosa26 lineage tracer) embedded in either wild-type UGSM or UGSM engineered to overexpress BMP5 (Figure 3E). After 90 days of growth under the kidney capsule, the PRU frequency averaged 1 in 7878 in normal UGSM and increased to 1 in 2059 in BMP5-expressing UGSM, an effect equivalent to the loss of Gata3 in KRT5+ basal cells (1 in 1,879) (Figure 1C). These grafts showed a formation of well-organized ductal structures including both basal and luminal lineages (Figure 3F and Figure 3—figure supplement 3). Hence, the exposure of basal stem/progenitor cells to increased BMP5 levels is sufficient to increase their regenerative potential. However, this experiment does not directly address whether BMP5 acts as a maintenance factor from the stromal mesenchyme or from within the basal cell compartment. To test this, we compared the regenerative potential of wild-type basal cells embedded in mesenchyme derived from either wild-type or Bmp5 animals (Figure 3E). This experiment revealed no significant difference in the PRU frequency or lineage potential of the grafts grown in presence or absence of mesenchymal Bmp5, indicating a role for BMP5 as a regulator of stem/progenitor cell homeostasis intrinsic to the basal cell layer.

Figure 3—figure supplement 3.

Allografts form well-organized ductal structures with both basal and luminal lineages.

Immunofluorescence analysis of lineage-specific markers KRT5 and KRT8/18 in indicated allografts. Lower magnification pictures of allograft from Figure 3D.

To directly assess the autonomous role of BMP5 in the basal compartment, we compared the regenerative potential of wild type or Bmp5 basal cells (expressing the constitutively active Rosa26 lineage tracer) embedded in wild-type UGSM cultures. After 90 days under the kidney capsule, the calculated PRU frequency averaged 1 in 7878 in the wild-type basal population but dramatically decreased to 1 in 20,175 in the absence of Bmp5 (Figure 3E). Together, these results demonstrate that BMP5 is critically required to sustain a full regenerative capacity in basal cell-derived allografts and identify BMP5 as an important regulator of basal stem/progenitor cells maintenance in the prostate.

BMP5 deficiency delays Pten-dependent tumor progression

One of the earliest and most frequent events in prostate cancer progression is the loss of the tumor suppressor PTEN (Abeshouse et al., 2015). Accordingly, Pten loss in the mouse prostate leads to carcinoma within 6–8 weeks (Mulholland et al., 2011; Trotman et al., 2003; Wang et al., 2003). Those tumors are castration-resistant, which mimics recurrent castration-resistant prostate cancer (CRPC) and are highly enriched in prostate stem cells (Wang et al., 2003). We previously showed that GATA3 is progressively lost in the prostate epithelium of Pten-deficient mice, while enforced GATA3 expression slows down tumor progression (Nguyen et al., 2013). In light of these results, we hypothesized that BMP5 signaling may contribute to the maintenance of Pten-deficient cancer stem cells.

To assess this possibility, we first performed a short-term organoid-forming assay using purified basal cells from Krt5 adult prostates. As expected, in vitro induction of the Cre recombinase by tamoxifen treatment led to an increase in organoid-forming potential over time (Figure 4A and Figure 4—figure supplement 1A). To test whether BMP signaling affects the self-renewal potential of cancer cells, we treated Pten-deficient basal prostate organoids with the inhibitory protein NOGGIN. Inhibition of BMP signaling affected their organoid-forming capacity, abrogating the long-term expansion in organoid cultures (Figure 4A and Figure 4—figure supplement 1A). This result suggests that the increased propagation potential of Pten-deficient prostate cancer stem/progenitor cells also requires BMP signaling.

Figure 4.

BMP inhibition reduces Pten-deficient propagation potential and inhibits skin and prostate tumor initiation.

(A) The aberrant organoid-forming capacity of Pten-deficient basal cells is abrogated by BMP inhibitor (NOGGIN) treatment. Cre activity was induced in vitro by treatment with hydroxy-tamoxifen for the first passage of organoid derived from basal cells of Krt5 mice. From passage 5, organoids were cultured in presence or absence of NOGGIN. Organoid-forming potential was assessed as in Figure 1A. (B) Krt5 tamoxifen-treated mice were injected with either K02288 or PBS for 4 weeks. (C–D) Representative histological sections of prostate tissue (C) and skin tissue (D) stained with H&E showing an absence of PIN and skin hyperplasia in K02288-treated as compared to mock-treated mice. (E) Kaplan-Meier skin tumor free survival curves of tamoxifen-induced Krt5 treated or not with K02288 (log-rank (Mantel-Cox) test; p<0.0001, n = 7 and n = 14, respectively). Tick-marks represent sacrificed animals. (F) Bmp5 loss rescues the aberrant organoid-forming capacity of Pten-deficient basal cells. Cre activity was induced in vivo by tamoxifen injection in adult mice 4 weeks prior to organoid propagation potential assessment. (G–L) Eight-week-old mice were treated with tamoxifen and sacrificed 4 weeks later. (H–I) Bmp5 loss and Gata3 overexpression rescues Pten-deficient prostate and skin hyperplasia. Shown are representative H&E pictures of prostate (H) and skin (I) tissues. (J) Kaplan-Meier skin tumor free survival curves of tamoxifen-induced Krt5, Krt5 and Krt5 mice (log-rank (Mantel-Cox) test; p<0.0001, n = 22, n = 17 and n = 9, respectively). Tick-marks represent sacrificed animals. (K–L) Representative FACS phenotype (K) and percentage of SCA1hi cells (L) in the luminal compartment as defined by Lin(CD45, CD31, TER119)-EpCAM+CD49fMed of total prostate (one-way ANOVA; ***p<0.0001). (M) Representative H&E pictures of prostate tissues of Krt5 and Krt5 mice sacrificed 9 weeks after tamoxifen treatment. See also Figure 4—figure supplements 1,2.

(A–B) Growth rate of cells upon organoid passage for the indicated genotypes and treatment calculated from nonlinear regression curve fit of data from Figure 4A and B, respectively (one-way ANOVA; *p<0.05, **p<0.005, ***p<0.0001). (C–D) Higher magnification H&E staining pictures of skin tissue from Figure 4D and I.

(A–B) BMPR inhibition affect the growth of Pten-deficient cell lines. Shown is the growth curve (A) and protein and phosphorylation levels of AKT (B) in CaP2 and CaP8 culture treated or not with K02288 at the indicated concentration (one-way ANOVA; p<0.0001).

Figure 4—figure supplement 1.

BMP inhibition corrects Pten-deficient tumor phenotypes.

(A–B) Growth rate of cells upon organoid passage for the indicated genotypes and treatment calculated from nonlinear regression curve fit of data from Figure 4A and B, respectively (one-way ANOVA; *p<0.05, **p<0.005, ***p<0.0001). (C–D) Higher magnification H&E staining pictures of skin tissue from Figure 4D and I.

Figure 4—figure supplement 2.

K02288 acts downstream of AKT pathway.

(A–B) BMPR inhibition affect the growth of Pten-deficient cell lines. Shown is the growth curve (A) and protein and phosphorylation levels of AKT (B) in CaP2 and CaP8 culture treated or not with K02288 at the indicated concentration (one-way ANOVA; p<0.0001).

To validate this result, we used the specific BMPR-SMAD1/5/8 signaling inhibitor K02288 on two Pten-deficient cell lines (CaP2 and CaP8). K02288 specifically abrogated culture growth without affecting cell viability or the expression or phosphorylation of the PTEN effector AKT (Figure 4—figure supplement 2A–B), indicating that K02288 acts downstream of AKT-mediated signaling.

We then tested BMP signaling inhibition in vivo by K02288 treatment of Krt5 adult mice for 30 days (at which point the mock-treated mice developed skin tumors and had to be sacrificed) (Figure 4B–E). Drug treatment led to a reduction of prostate lesions in comparison to mock-treated mice that had accumulated prostatic intraepithelial neoplasia (PIN) at this stage (Figure 4C). Interestingly, K02288 treatment additionally led to a marked reduction of the severity of skin lesions derived from KRT5-positive basal cells and greatly delayed the onset of tumor growth (Figure 4D-E and Figure 4—figure supplement 1A), indicating that the promotion of Pten-deficient tumor expansion by BMP signaling is not limited to the prostate. To determine whether Pten-deficient neoplasia was specifically dependent on the BMP5 ligand, we inactivated both Pten and Bmp5 in KRT5-positive basal cells of the prostate and skin (Figure 4G). Loss of Bmp5 impaired the aberrant organoid-forming capacity of Pten-deficient prostate cells (Figure 4F and Figure 4—figure supplement 1B), as well as the increase in numbers of aberrant SCA1hi luminal progenitor cells (Figure 4K–L). Strikingly, Krt5 mice showed a reduction in the formation of prostate and skin hyperplasia as well as a delay in tumor growth onset similar to animals treated with K02288 (Figure 4H,I,J and Figure 4—figure supplement 1D, E, F). This correction of prostate tumor phenotype by Bmp5 loss was still observed 9 weeks after Cre induction (Figure 4M). Interestingly, prostate and skin tumor phenotypes could also be dampened by overexpression of GATA3 in these tissues (Figure 4H–L) indicating that both Gata3 and BMP5 are key players in Pten-deficient cancer progression. These results additionally raise the possibility to use a small inhibitor against BMP signaling as a treatment against PTEN-deficient cancer progression both in the prostate and in the skin.

Discussion

In recent years, the presence of adult prostate stem cells has been reported in both the basal and luminal compartments (Choi et al., 2012; Goldstein et al., 2010; Liu et al., 2011; Wang et al., 2013). However, the question of how these cells regulate the balance between long-term maintenance and differentiation remained unanswered. Here, we tackle this question in adult basal stem/progenitor cells. We show that prostate basal cells devoid of the transcription factor Gata3 increase their self-renewal capacity in long-term propagation assays and further identify BMP5 as a crucial mediator of this activity. Using gain and loss-of-function approaches, we demonstrate that BMP signaling is required for sustained stem/progenitor cell propagation within the basal cell compartment. Using a mouse model of prostate cancer highly enriched in cancer stem cells, we finally show that inhibition of BMP5 signaling is sufficient to delay cancer progression from basal cells in the prostate and in the skin. Together, these observations demonstrate that BMP signaling promotes basal stem cell self-renewal, while GATA3 counteracts this activity to regulate the basal stem cell pool.

BMP signaling has been linked to stem cell maintenance in a number of organisms, often acting as a niche factor (Chen et al., 2011; Oshimori and Fuchs, 2012; Tian and Jiang, 2014). However, whether BMP signaling promotes stem cell self-renewal or quiescence is context-dependent (Badeaux et al., 2013; Chen et al., 2011; Chung et al., 2018; Genander et al., 2014; Kangsamaksin and Morris, 2011; Lewis et al., 2014; Li et al., 2012; Oshimori and Fuchs, 2012; Tadokoro et al., 2016; Tian and Jiang, 2014). Our results in the prostate clearly identify a role for BMP signaling in promoting the long-term maintenance of prostate stem/progenitor cells, that appears to be intrinsic to the basal cell compartment as opposed to an exogenous niche factor. In support of this, Gata3 deficiency leads to an increase in BMP5 expression specifically in basal cells, while Gata3-deficient prostate organoids show a strong stem/progenitor cell amplification phenotype despite being devoid of stromal niche. This basal cell amplification can be blocked by BMP inhibitor treatment and is blunted in Bmp5-deficient cells indicating that BMP signaling is required for stem/progenitor cell amplification. An alternative possibility would be that BMP5 acts as a niche factor from luminal cells. However, Gata3 inactivation by luminal-specific Krt8 failed to increase the organoid-forming potential, arguing against this possibility. Importantly, while BMP5-overexpressing mesenchyme could enhance basal stem cell activity in renal allograft assay, Bmp5-deficient mesenchyme remained competent in stem cell derived allograft growth. In contrast, Bmp5-deficient basal cells had a blunted regenerative potential in the presence of wild-type mesenchyme. Hence, increased exposure to BMP5 expressed from the UGSM can support basal stem/progenitor cells, but ultimately it is BMP5 expression from the basal compartment that is critical to basal stem cell homeostasis. The intrinsic role of BMP5 in the basal cell compartment is further supported by the identification of Bmp5 as one of the most highly expressed genes in a prostate basal stem cell population defined by s-SHIP1 gene expression (Brocqueville et al., 2016). The molecular mechanism by which GATA3 modulates Bmp5 in basal cells remains elusive. However, the direct binding of GATA3 to the Bmp5 locus in mouse and human, combined with the fact that GATA3 is known to interact with repressive chromatin modifier complexes (Tremblay et al., 2018) suggests a molecular mechanism to be explored further.

Taken together, these results favor a model by which GATA3 regulates stem/progenitor cell long-term maintenance by modulating the expression levels of the autocrine stem cell factor BMP5.

PTEN mutation is one of the earliest and most frequent alterations in prostate cancer (Abeshouse et al., 2015). Accordingly, Pten-deficient prostates develop castration-resistant prostate cancer (Mulholland et al., 2011; Wang et al., 2003) that are highly enriched in cancer stem cells (this report) (Goldstein et al., 2010; Mulholland et al., 2009; Wang et al., 2014a). To validate whether the increase in BMP signaling was involved in Pten-deficient tumors, we treated Pten-deficient prostate organoids and mouse prostate tumors with a selective inhibitor against BMP signaling, which resulted in blunted organoid passaging potential and a delay in tumor progression in vivo. These results are in line with a previous report showing that loss of TGFβ signaling has been linked to prostate cancer progression in Pten-deficient tumor, in part through upregulation of BMP signaling (Zhao et al., 2018). Here, we identify the ligand BMP5 as prime driver of Pten-deficient cancer progression. It is possible that other BMPs are involved in tumor progression that would also be blocked by small molecule-mediated inhibition of BMP signaling. For example, BMP6, a member of the BMP5/6/7 subfamily, is upregulated in prostate cancer and contributes to cancer progression (Darby et al., 2008). Whether BMP5 and BMP6 have overlapping or distinct roles remains to be determined. However, our results clearly demonstrate that Bmp5 inactivation alone is sufficient to significantly delay cancer development with an efficiency similar to BMP pathway inhibition, identifying this ligand as a central player in the process.

In this study, we specifically inactivated Pten in basal cells, that are known to transition to a luminal fate and generate carcinoma under stress conditions (Wang et al., 2014b; Wang et al., 2013). Our results demonstrate that dampening the BMP-regulated cancer stem cell pool in basal cells is an effective strategy to reduce the tumorigenic potential of this population. Whether or not the GATA3-BMP axis is also effective in luminal cells of the prostate is still unclear. Our results suggest that the upregulation of BMP5 by Gata3 loss occurs mostly in basal cells. However, the fact that Gata3 could modulate prostate cancer progression in Pbsn-Cre; Pten-deficient mice (Nguyen et al., 2013) known to develop largely from luminal cells raise the possibility of a role in luminal cells. One possibility would be that Gata3 acts through the regulation of other BMP family members to achieve a similar role in different cell types.

Remarkably, as skin cancer can originate from KRT5-positive basal stem cells (Suzuki et al., 2003), the strategy of BMP inhibition proved also effective in this tissue. BMP5 was previously reported to play a role in non-cancerous keratinocytes where Bmp5 inactivation reduces the number of label-retaining cells, while exogenous BMP5 increased colony formation (Badeaux et al., 2013; Kangsamaksin and Morris, 2011). These results support a role for BMP5 in stem cell maintenance, comparable to the one described here. The effective activity of BMP inhibition in both tissues further raise the prospect of a therapeutic approach in the treatment of Pten-deficient tumors.

Materials and methods

Mice

All experimental mice were kept in a C57BL/6 background. Pbsn-Cre (Wu et al., 2001), Gata3 (Grote et al., 2006), Gata3 (kind gift from Dr. Busslinger, IMP, Vienna; see Figure 2—figure supplement 3 for generation strategy), Rosa26 (Wood et al., 2016), Krt5CreERT2 (Van Keymeulen et al., 2011), Krt8CreERT2 (Choi et al., 2012), Rosa26LstopLTdTomato (Madisen et al., 2010), Pten (Trotman et al., 2003) and Bmp5SE (Kingsley et al., 1992) mice were described previously. Constitutively active Rosa26LTdTomato were generated from female Pbsn-Cre mice which express Cre in the germline and backcrossed to C57BL/6 to eliminate the Cre allele. Immunodeficient SCID-beige mice were obtained from Charles River and kept in pathogen-free conditions. Except stated otherwise, all experiments were done using 8-week-old adult mice. In vivo CreERT2 induction was done by intraperitoneal injection of three doses of 3 mg of tamoxifen dissolved in corn oil. Mice were injected daily intraperitoneally with either mock or 500 mg of K02288 (AdooQ Bioscience) dissolved in PBS containing 10% DMSO. Kaplan–Meier skin tumor‐free survival curves were obtained using Prism 6.0 software (GraphPad). All animal procedures were approved by McGill University Animal Care Committee according to the Canadian Council on Animal Care guidelines for use of laboratory animals in biological research.

Figure 2—figure supplement 3.

Generation strategy of Gata3 allele.

FACS sorting and analysis

Whole prostate tissue from a minimum of three mice were dissected and pooled in cold PBS 2% FBS, minced and digested at 37°C for 2 hr in DMEM 5% FBS 1X Collagenase/hyaluronidase (Stemcell Technologies) for 5 min in 0.25% trypsin/EDTA and followed by 10 min in dispase II (5 U/ml) and DNase I (0.1 mg/ml) (Roche). All solution contained 10 μM of Rock inhibitor Y-27632 (ApexBio). The digested cells were passed through a 27G needle and filtered through a 70 μm cell strainer. Cell staining was done on ice for 30 min using human IgG blocking solution and antibodies CD45 (30-F11), TER119 (TER-119), CD31 (MEC13), CD49f (GoH3), EpCAM (G8.8), SCA1 (D7) (all from Biolegend) and TROP2 (from R and D). Dead cells were excluded by fixable Viability dye (eBioscience) staining. FACS analysis and sorting was performed on a BD Fortessa and Aria Fusion apparatus (BD Biosciences). Data was analyzed using DIVA (BD Biosciences) and FlowJo softwares.

Short-term organoid propagation assay

LIN-SCA1+CD49F+EPCAM+TROP2+ basal cells were sorted from whole prostate and plated in advanced DMEM/F12 (ThermoFisher) 50% matrigel (Corning) around the rims of 12-well plates. Organoids were grown for 7 days in modified organoid media composed of WIT basic media (Cellaria) supplemented with 10 ng/ml EGF, 25 ug/ml BPE, 1X B27 (Life Technologies) and 10 µM Y-27632. Pictures of organoid culture were taken with an AxioObserver (Zeiss) and organoid size was analyzed using ImageJ (Fiji) software. Passaging of organoids was done by digestion with dispase for 1 hr at 37°C and Trypsin 0.25% EDTA for 5 min, syringe trituration and replating in 50% matrigel. Viable cells from dissociated organoids were counted using TC10 cell counter (Biorad) with trypan blue and replated at a concentration of 105 per wells. In vitro CreERT2 induction was done by treatment of organoid with 500 ng/ml 4-hydroxy-tamoxifen (4-OHT). Media supplemented with either exogenous 50 ng/ml of recombinant hBMP5 (Aviscera Bioscience), 180 ng/ml recombinant hNOGGIN (RayBiotech) or 10 μM of K02288 inhibitor (AdooQ Bioscience) was changed every other day. Organoid differentiation was done by supplementing culture media with 10−8M 5α-dihydrotestosterone (DHT, Steraloids). Electroporation of shRNAs either scrambled or against Bmp5 (MISSION pLKO.1-Puro shRNA; Sigma-Aldrich) was done on single cells dissociated from passage one organoid using P1 Primary Cell 4D-Nucleofector Kit (Bioscience) using program EL-110 and selected using 0.5 μg/ml of puromycin. Growth rate of cells upon organoid passage was calculated using nonlinear regression curve fitting and significance between genotype was assessed by one-way ANOVA using Prism 6.0 software (GraphPad).

Allograft/limiting dilution assay

Urogenital sinus mesenchyme (UGSM) culture were described previously (Xin et al., 2003), briefly the urogenital sinus from wild type or Bmp5 embryos between E14.5 to E16.5 was digested in 1% trypsin in HBSS for 90 min at 4°C. Mesenchyme was separated from the epithelium and digested in 0.2% collagenase A for 1 hr at 37°C. Single cells were plated on fibronectin treated plastic in DMEM medium 5% FBS 5% Nu serum 1% Non-Essential Amino Acids (NEAA) 1% glutamine and 1 nM 5α-dihydrotestosterone (DHT, Steraloids). UGSM overexpressing BMP5, was generated by infection with lentiviral particles from either empty or BMP5 expressing pLenti plasmid and subjected to selection with puromycin. Subrenal regeneration assays have been previously described (Doles et al., 2005). Briefly, sorted TdTomato+ basal cells from a pool of a minimum of three mice from either Pbsn-Cre Rosa26LstopLTdtomato, Pbsn-Cre Gata3LstopLTdtomato, Krt5LstopLTdtomato, constitutively active Rosa26TdTomato or Bmp5SE/SERosa26TdTomato genotype were mixed with 105 UGSM cells of the indicated genotype in rat collagen I (Corning) and implanted under the kidney capsule of SCID-beige recipient mice. Growth of prostate tissue grafts was stimulated by subcutaneous implantation of silastic testosterone implant of 25 mg every 3 weeks for 90 days. An outgrowth was defined as an epithelial TdTomato+ fluorescent structure comprising ducts. A minimum of four replicates was done per dilution and using Poisson distribution, Prostate reconstituting unit (PRU) frequency was calculated for basal cells from the whole prostate following limiting dilution transplantation using L-calc software (Stem cell technologies). Student's t-tests were performed for statistical analysis between two groups.

Microscopy and image analysis

Immunofluorescence and H&E staining were performed on PFA-fixed organoid or tissue embedded in paraffin or on freshly frozen tissue embedded in OCT, sectioned to obtain 4- and 10-μm-thick sections as described previously, respectively (Nguyen et al., 2013). Staining was done overnight using the following primary antibodies: KRT5 (1:500, Biolegend #905901), KRT8/18 (1:200, Fitzgerald #20R-CP004), GATA3 (1:100, SantaCruz #sc-9009) and Ki67 (1:200, eBioscience #14-5698-82). TUNEL staining was done using the In Situ Cell Death Detection Kit (Roche), following manufacturer’s protocol. Immunofluorescence images were acquired using either an LSM710, LSM780 or LSM800 confocal microscope (Zeiss). H&E images were scanned on an AperioScanScope AT, whereas brightfield images were taken with an Axio Observer Z1 (Zeiss).

RNA isolation, quantitative RT-PCR and transcriptomic profiling

Total RNA was extracted from sorted population of prostate cells or organoid cultures using a RNeasy micro kit (Qiagen). To increase the proportion of stem/progenitor cells in the population, organoids were harvested after 4 days in culture (instead of 7). RNA was reverse transcribed with MMLV (Invitrogen) according to manufacturer’s procedures. Real-time quantitative PCR was performed using Green-2-go mastermix (BioBasic Inc) on Realplex2 Mastercycler (Eppendorf). Total RNA was isolated and sequenced from day 4 organoids of wild type and Pbsn-Cre Gata33 at passages 0, 2, 3 and 4. Sequencing libraries were prepared by Genome Quebec Innovation Centre (Montreal, Canada), using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina TS-122–2301, San Diego, CA) following depletion or ribosomal and fragmented RNA. The libraries were sequenced using the Illumina HiSeq 2000 sequencer, 100 nucleotide paired-end reads, generating approximately 60 million reads per sample. The sequencing reads were pseudo aligned to the mouse reference genome (mm10) using default parameters in Kallisto (Bray et al., 2016). Transcripts were annotated using Ensembl release 89. Abundance estimate and bootstrap values generated by Kallisto were used for expression quantification (Bray et al., 2016). Differential expression testing was performed to identify genes differently expressed between genotypes and passages with Sleuth using either a likelihood ratio test (LRT) or Walds test (WT) (Pimentel et al., 2017) from Sleuth package. Maps of sequencing reads were generated using R and the ggplot2 package (version 1), and bedtools (Quinlan and Hall, 2010). Analysis of Gata3-deletion of exon four by Pbsn-Cre was done by mapping raw reads to the Gata3 genomic locus using the bedtools package. Heatmap of normalized gene expression (Average = 0; variance = 1) were generated using matrix2png program (Pavlidis and Noble, 2003). All raw and processed transcriptome data are available from NCBI GEO (accession GSE155289).

Biotin-chromatin immunoprecipitation pulldown

Prostate tissue from Gata3 mice were minced down and crosslinked for 10 min with 1% formaldehyde. Formaldehyde was quenched by addition of 0.125 M glycine. Fixed cells were pelleted by centrifugation, washed twice in cold phosphate-buffered saline, then washed once in Triton buffer for 15 min (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and once in NaCl buffer for 15 min (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Cells were pelleted, resuspended in RIPA buffer (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate) and sonicated (7 × 10 s bursts) to make soluble chromatin ranging in size from 500 to 1000 bp. Cellular debris were removed by centrifugation (16,000 × g for 10 min), and protein concentrations were determined by Bradford staining. Crosslinked extracts were subjected to BioChIP pulldown using streptavidin conjugated beads or IgG control beads. Bound extracts were sequentially washed twice with 1 ml of RIPA buffer, twice with 1 ml of LiCl buffer (10 mM Tris-HCl, pH 8.0, 250 mMLiCl, 1 mM EDTA, 1% Nonidet P-40, 1% deoxycholate) and twice with 1 ml of TE buffer. Chromatin samples were then eluted by heating for 15 min at 65°C in 300 μl of elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). After centrifugation, supernatants were diluted by addition of 300 μl of TE buffer and heated overnight at 65°C to reverse cross-links. RNA and proteins were sequentially degraded by addition of 30 μg of RNase A for 30 min at 37°C, and 120 μg of proteinase K for 2–3 hr at 37°C. DNA was phenol/chloroform-extracted and ethanol-precipitated in the presence of 10 μg of tRNA as a carrier. Amplification of the indicated locus was done by quantitative PCR using Green-2-go mastermix (BioBasic Inc) on Realplex2 Mastercycler (Eppendorf).

Cell lines and western blot

CaP2 and CaP8 murine prostatic cells were provided by Dr. Hong Wu (UCLA) and maintained as described (Jiao et al., 2007) and confirmed to be mycoplasma negative by PCR. Growth curves of treated cells with indicated amount of K02288 were obtained using phase object confluence as monitored every 2 hr by IncuCyte S3 live cell imaging (Essen Bioscience). Significance between genotype was assessed by one-way ANOVA using Prism 6.0 software (GraphPad). Protein extracts were prepared after 1 hr treatment with K02288 using RIPA lysis buffer (10 mM Tris–HCl (pH 8.0), 140 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulphate, 0.1% deoxycholate, 1 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Roche). Proteins were immobilized on Immobilon FL PVDF membranes (Millipore) and probed with the following antibodies diluted in Odyssey blocking buffer (LI-COR): anti-pAKT S473 (1:500, CST), anti-AKT (1:500, CST), and anti-α-TUBULIN (DM1A) (1:2000, Sigma). Rabbit and mouse IR dye secondary antibodies (LI-COR Biosciences) were used at 1:10 000. Blots were scanned with the Odyssey imaging system (LI-COR).

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Through a combination of genetic and pharmacologic approaches, your work convincingly demonstrates a role of GATA3 in regulating self-renewal of prostate basal epithelial cells, largely through transcriptional repression of BMP5. Interestingly, this GATA3/BMP5 regulatory circuit is restricted to basal cells and is not present in luminal cells. A second finding is the importance of BMP5 in the oncogenic phenotype of Pten loss, based on reduced levels of prostate intraepithelial neoplasia (PIN) in the setting of genetic BMP5 deletion or pharmacologic inhibition. An important remaining question for future work is whether this BMP5 dependency is a consequence of perturbation of the GATA3/BMP5 regulatory circuit (in basal cells) or through BMP5 expression in luminal cells.

Decision letter after peer review:

Thank you for submitting your article "Regulation of stem/progenitor cell maintenance by BMP5 in prostate homeostasis and cancer initiation" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Charles Sawyers as Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Dean Tang.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The authors provide evidence that implicates BMP5 (positively) and Gata3 (negatively) in regulating prostate stem/progenitor cells in the context of both homeostasis and tumor development. This group has been investigating Gata3 in both normal prostate and prostate cancer (e.g., Nguyen et al., 2013; Shafer et al., 2017). Here, the authors implicate BMP5 as a key regulator of basal prostate stem cell homeostasis. They provide data that Bmp5 gene inactivation or chemical-mediated inhibition of Bmp signaling delays prostate and skin tumorigenesis initiated by Pten loss.

Essential revisions:

1) The use of Noggin to implicate BMP5 is not definitive because Noggin is not specific for Bmp5 inhibition. We would like to see confirmatory data with Bmp5 shRNA to corroborate the role of the Gata3-Bmp5 signaling axis in organoid formation.

2) Figures 3C and 4H, the results convincingly demonstrate that decreased Bmp5 signaling attenuated prostate regeneration and PIN progression. But these data need to be better connected to the GATA3-Bmp5 signaling axis studies presented in the first part of the study (Figures 1 and 2). For example, do Gata3 KO basal cells possess a higher regenerative capacity in vivo, like the basal cells stimulated with ectopic Bmp5?

In addition, since upregulation of Bmp5 only happened when Gata3 was deleted in basal cells (SF2a), the authors should comment (with additional data or in the Discussion) on whether Gata3 KO/overexpression will impact PIN progression in the Keratin8-CreER-Pten (luminal Cre) model? This is an important point regarding whether the Gata3-Bmp5 signaling axis is likely to play a critical role in prostate cancer progression.

Essential revisions:

1) The use of Noggin to implicate BMP5 is not definitive because Noggin is not specific for Bmp5 inhibition. We would like to see confirmatory data with Bmp5 shRNA to corroborate the role of the Gata3-Bmp5 signaling axis in organoid formation.

In support of a specific implication of Bmp5, we added several new results showing that Bmp5 inhibition or loss by either inhibitor treatment, knockout, knockdown strategy impairs the propagation potential of wild type as well as Gata3-deficient organoid cultures:

First, we treated our culture with a selective inhibitor of BMPR-SMAD1/5/8 signaling, K02288, which validate the effect seen by Noggin treatment (Figure 3B, Figure 3—figure supplement 2B).

Second, we created a new line of mice where both Bmp5 and Gata3 can be knocked-out in KRT5+ basal cells. We see that Bmp5 mutation impaired the increased organoid-forming capacity of Gata3-deficient cells which clearly demonstrate that Gata3-deficient phenotype is specifically dependent on the BMP5 ligand (Figure 3C, Figure 3—figure supplement 2C).

Finally, we also provide evidences that the acute loss of Bmp5 using shRNA against Bmp5 (knockdown) showed a similar effect, inhibiting the passaging capacity of both wild type and Gata3-deficient organoids (Figure 3D, Figure 3—figure supplement 2D). Together, these results clearly confirm that the increased propagation potential of Gata3-deficient basal cells requires BMP signaling driven by the BMP5 ligand.

2) Figures 3C and 4H, the results convincingly demonstrate that decreased Bmp5 signaling attenuated prostate regeneration and PIN progression. But these data need to be better connected to the GATA3-Bmp5 signaling axis studies presented in the first part of the study (Figures 1 and 2). For example, do Gata3 KO basal cells possess a higher regenerative capacity in vivo, like the basal cells stimulated with ectopic Bmp5?

In the revised version we added several new results showing that the Gata3-Bmp5 axis is link to an increase in regenerative potential and correction of prostate cancer progression.

First, as presented before in Figure 1C, Gata3 loss in sorted basal cells using the Pb driver show a higher regenerative capacity compared to wild type cells in vivo.

Second, we provide new results showing that the specific loss of Gata3 in basal cells using Krt5 driver (PRU frequency of 1 in 1,879, Figure 1C) lead to a 4-fold increase in regenerative potential similar to basal cells stimulated with ectopic BMP5 (PRU frequency of 1 in 2,059, Figure 3E).

Third, we provide new organoid culture results showing the effect of Bmp5 inhibition or loss by either inhibitor treatment, knockout, knockdown strategy on the propagation potential of wild type as well as Gata3-deficient organoid cultures (Figure 3B-D, Figure 3—figure supplement 2B-D, see comment above).

Finally, we created a new line of mice where GATA3 is overexpress together with Pten loss specifically in basal cells (Krt5). Similar to the loss of Bmp5, overexpression of Gata3 corrects Pten-deficient tumor phenotype in both the prostate and the skin (Figure 4H-L, Figure 4—figure supplement 2D) indicating that both Gata3 and BMP5 are key players in Pten-deficient cancer progression.

In addition, since upregulation of Bmp5 only happened when Gata3 was deleted in basal cells (SF2a), the authors should comment (with additional data or in the Discussion) on whether Gata3 KO/overexpression will impact PIN progression in the Keratin8-CreER-Pten (luminal Cre) model? This is an important point regarding whether the Gata3-Bmp5 signaling axis is likely to play a critical role in prostate cancer progression.

We deliberately decided to focus on basal cells based on the basal-specific response of Bmp5 to the loss of Gata3 (Figure 2—figure supplement 2A). By covering luminal cells, our manuscript would have lost in cohesion and depth. In support of a role for Bmp5 in the luminal compartment, we treated Krt8 mice with the selective inhibitor of BMPR-SMAD1/5/8 signaling, K02288, for 72 days after tamoxifen induction. Our preliminary results show a correction of prostate phenotype (see Author response image 1). These results suggest strongly that GATA3 and BMP5 also play a role in luminal cells but this will have to be addressed in a separate manuscript and include a different set of experiments. We therefore added a section about this in the Discussion as suggested.

Author response image 1.

Krt8 tamoxifen-treated mice were injected with either K02288 or PBS for 10 weeks.

Showed are representative histological sections of prostate tissue stained with H&E showing an absence of PIN in K02288-treated as compared to mock-treated mice.

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (Mus musculus)	ARR2-PB-Cre (Pbsn-Cre), C57BL/6 (Mus musculus)	Wu et al., 2001	RRID:IMSR_JAX:026662	(Mus musculus; male)
Strain, strain background (Mus musculus)	Gata3flox, C57BL/6 (Mus musculus)	Grote et al., 2006		(Mus musculus; male)
Strain, strain background (Mus musculus)	Gata3GFP, C57BL/6 (Mus musculus)	Grote et al., 2006		(Mus musculus; male)
Strain, strain background (Mus musculus)	Rosa26GATA3, C57BL/6 (Mus musculus)	Grote et al., 2006		(Mus musculus; male)
Strain, strain background (Mus musculus)	Gata3bio, C57BL/6 (Mus musculus)	Dr. Busslinger, IMP, Vienna		(Mus musculus; male)
Strain, strain background (Mus musculus)	Rosa26BirA, C57BL/6 (Mus musculus)	Wood et al., 2016	RRID:IMSR_JAX:010920	(Mus musculus; male)
Strain, strain background (Mus musculus)	Krt5CreERT2, C57BL/6 (Mus musculus)	Van Keymeulen et al., 2011	RRID:IMSR_JAX:029155	(Mus musculus; male)
Strain, strain background (Mus musculus)	Krt8CreERT2, C57BL/6 (Mus musculus)	Choi et al., 2012		(Mus musculus; male)
Strain, strain background (Mus musculus)	Rosa26LstopLTdTomato, C57BL/6 (Mus musculus)	Madisen et al., 2010	RRID:IMSR_JAX:007909	(Mus musculus; male)
Strain, strain background (Mus musculus)	Ptenflox , C57BL/6 (Mus musculus)	Trotman et al., 2003		(Mus musculus; male)
Strain, strain background (Mus musculus)	Bmp5SE, C57BL/6 (Mus musculus)	Kingsley et al., 1992	RRID:IMSR_JAX:000056	(Mus musculus; male)
Strain, strain background (Mus musculus)	SCID-beige, C57BL/6 (Mus musculus)	Charles River		(Mus musculus; male)
Cell line (Mus musculus)	CaP2 (Mus musculus)	Jiao et al., 2007		male prostate
Cell line (Mus musculus)	CaP8 (Mus musculus)	Jiao et al., 2007		male prostate
Transfected construct (Human)	BMP5_TRC3 LentiORF puromycin V5 (pLX317) (human)	Sigma-Aldrich	TRCN0000472902	mouse UGSM
Transfected construct (Human)	TRC3 LentiORF puromycin V5 (pLX317) empty (human)	Sigma-Aldrich		mouse UGSM
Antibody	CD45 (30-F11; rat monoclonal)	Biolegend		(1/500)
Antibody	TER119 (TER-119; rat monoclonal)	Biolegend		(1/1,000)
Antibody	CD31 (MEC13; rat monoclonal)	Biolegend		(1/1,000)
Antibody	CD49f (GoH3; rat monoclonal)	Biolegend		(1/2,000)
Antibody	EpCAM (G8.8; rat monoclonal)	Biolegend		(1/500)
Antibody	SCA1 (D7; rat monoclonal)	Biolegend		(1/500)
Antibody	TROP2 (goat polyclonal)	R&D		(1/100)
Antibody	KRT5 (chicken polyclonal)	Biolegend	905901	(1/500)
Antibody	KRT8/18 (guinea pig polyclonal)	Fitzgerald	20R-CP004	(1/200)
Antibody	GATA3 (H-48; rabbit polyclonal)	SantaCruz	sc-9009	(1/100)
Antibody	Ki67 (SolA15, rat monoclonal)	eBioscience	14-5698-82	(1/200)
Antibody	pAKT S473 (rabbit monoclonal)	CST	4060	(1/500)
Antibody	AKT (rabbit polyclonal)	CST	9272	(1/500)
Antibody	α-TUBULIN (DM1A; mouse monoclonal)	Sigma	T9026	(1/2000)
Antibody	IRDye 800CW Donkey anti-Rabbit IgG	licor	926-32213	(1/10,000)
Antibody	IRDye 680RD Donkey anti-Mouse IgG	licor	926-68072	(1/10,000)
Recombinant DNA reagent	MISSION pLKO.1-Puro shRNA scrambled (mouse)	Sigma-Aldrich
Recombinant DNA reagent	MISSION pLKO.1-Puro shRNA Bmp5 (mouse)	Sigma-Aldrich	TRCN0000065609
Recombinant DNA reagent	MISSION pLKO.1-Puro shRNA Bmp5 (mouse)	Sigma-Aldrich	TRCN0000065610
Recombinant DNA reagent	MISSION pLKO.1-Puro shRNA Bmp5 (mouse)	Sigma-Aldrich	TRCN0000065611
Sequence-based reagent	Bmp5_Fw	Sigma-Aldrich		TTACTTAGGGGTATTGTGGGCT
Sequence-based reagent	Bmp5_Rv	Sigma-Aldrich		CCGTCTCTCATGGTTCCGTAG
Sequence-based reagent	Gata3_Fw	Sigma-Aldrich		GTGGTCACACTCGGATTCCT
Sequence-based reagent	Gata3_Rv	Sigma-Aldrich		GCAAAAAGGAGGGTTTAGGG
Sequence-based reagent	Krt5_Fw	Sigma-Aldrich		GAGATCGCCACCTACAGGAA
Sequence-based reagent	Krt5_Rv	Sigma-Aldrich		TCCTCCGTAGCCAGAAGAGA
Sequence-based reagent	Krt14_Fw	Sigma-Aldrich		CCTCTGGCTCTCAGTCATCC
Sequence-based reagent	Krt14_Rv	Sigma-Aldrich		TGAGCAGCATGTAGCAGCTT
Sequence-based reagent	Krt18_Fw	Sigma-Aldrich		ACTCCGCAAGGTGGTAGATGA
Sequence-based reagent	Krt18_Rv	Sigma-Aldrich		TCCACTTCCACAGTCAATCCA
Sequence-based reagent	Krt8_Fw	Sigma-Aldrich		CAAGGTGGAACTAGAGTCCCG
Sequence-based reagent	Krt8_Rv	Sigma-Aldrich		CTCGTACTGGGCACGAACTTC
Sequence-based reagent	Trp63_Fw	Sigma-Aldrich		AGCAGCAAGTATCGGACAGC
Sequence-based reagent	Trp63_Rv	Sigma-Aldrich		CGTCTCACGACCTCTCACTG
Sequence-based reagent	Ly6a_Fw	Sigma-Aldrich		CCATCAATTACCTGCCCCTA
Sequence-based reagent	Ly6a_Rv	Sigma-Aldrich		GGCAGATGGGTAAGCAAAGA
Sequence-based reagent	Itga6_Fw	Sigma-Aldrich		CGCTGCTGCTCAGAATATCA
Sequence-based reagent	Itga6_Rv	Sigma-Aldrich		AAGAACAGCCAGGAGGATGA
Sequence-based reagent	Epcam_Fw	Sigma-Aldrich		GCTGTCATTGTGGTGGTGTC
Sequence-based reagent	Epcam_Rv	Sigma-Aldrich		CACGGCTAGGCATTAAGCTC
Sequence-based reagent	Ppia_Fw	Sigma-Aldrich		GTGCCAGGGTGGTGACTTTACACG
Sequence-based reagent	Ppia_Rv	Sigma-Aldrich		TCCCAAAGACCACATGCTTGCCA
Sequence-based reagent	Bactin_Fw	Sigma-Aldrich		TTGCTGACAGGATGCAGAAGGAGA
Sequence-based reagent	Bactin_Rv	Sigma-Aldrich		ACTCCTGCTTGCTGATCCACATCT
Sequence-based reagent	Bmp5_prom_Fw-8450	Sigma-Aldrich		TCGGGTGGACCAGATTTAAG
Sequence-based reagent	Bmp5_prom_Rv-8390	Sigma-Aldrich		CAGCCATTCACGAAGTTCTCT
Sequence-based reagent	Bmp5_prom_Fw-5942	Sigma-Aldrich		TGAAAGTGGAGATGGGGAAG
Sequence-based reagent	Bmp5_prom_Rv-5827	Sigma-Aldrich		CCCAGTTTTGGAGGTTCAGA
Sequence-based reagent	Bmp5_prom_Fw+11268	Sigma-Aldrich		AAAGGGAAAAGTGCTCACCA
Sequence-based reagent	Bmp5_prom_Rv+11312	Sigma-Aldrich		TCCTCCCTCAGCTCAAAGAA
Sequence-based reagent	Bmp5_prom_Fw+11859	Sigma-Aldrich		TTGGAAGAGTTCCGATGAGG
Sequence-based reagent	Bmp5_prom_Rv+11947	Sigma-Aldrich		CAGAGTGGGTGGCAACTTCT
Sequence-based reagent	Bmp5_prom_Fw+24561	Sigma-Aldrich		GTGAGGTGGCTCAGCATGTA
Sequence-based reagent	Bmp5_prom_Rv+24607	Sigma-Aldrich		CCAGGGATGGATCTCAGGT
Sequence-based reagent	Cyp19a1_prom-322_Fw	Sigma-Aldrich		GCAAATGCTGCTGATGAAAT
Sequence-based reagent	Cyp19a1_prom-207_Rv	Sigma-Aldrich		ACCTTATCATCTCGCCCTTG
Sequence-based reagent	Cdh1_prom-175_Fw	Sigma-Aldrich		GAACGACCGTGGAATAGGAA
Sequence-based reagent	Cdh1_prom-98_Rv	Sigma-Aldrich		TCCTCCACCCCTGTCTGTAG
Sequence-based reagent	R26wt_geno_Fw	Sigma-Aldrich		AAGGGAGCTGCAGTGGAGTA
Sequence-based reagent	R26wt_geno_Rv	Sigma-Aldrich		CCGAAAATCTGTGGGAAGTC
Sequence-based reagent	R26Tomato_Fw	Sigma-Aldrich		CCCCGTAATGCAGAAGAAGA
Sequence-based reagent	R26Tomato_Rv	Sigma-Aldrich		GAGGTGATGTCCAGCTTGGT
Sequence-based reagent	Bmp5wt_geno_Fw	Sigma-Aldrich		TAAGGACAAGGGAAACCCTC
Sequence-based reagent	Bmp5SE_geno_Fw	Sigma-Aldrich		TAAGGACAAGGGAAACCCTT
Sequence-based reagent	Bmp5_geno_Rv	Sigma-Aldrich		GAACCATTTCACCAGCTCCT
Sequence-based reagent	Rosa26Gata3-wt_geno_Fw	Sigma-Aldrich		AAAGTCGCTCTGAGTTGTTAT
Sequence-based reagent	Rosa26Gata3_geno_Rv	Sigma-Aldrich		GCGAAGAGTTTGTCCTCAACC
Sequence-based reagent	Rosa26wt_geno_Rv	Sigma-Aldrich		GGAGCGGGAGAAATGGATATG
Sequence-based reagent	Gata3_flox_geno_Fw	Sigma-Aldrich		GTCAGGGCACTAAGGGTTGTT
Sequence-based reagent	Gata3_flox_geno_Rv	Sigma-Aldrich		TGGTAGAGTCCGCAGGCATTG
Sequence-based reagent	Gata3GFP_geno_Fw	Sigma-Aldrich		GGCCTACCCGCTTCCATTGCT
Sequence-based reagent	Gata3GFP_geno_Rv	Sigma-Aldrich		TATCAGCGGTTCATCTACAGC
Sequence-based reagent	Pten_flox-wt_geno_Fw	Sigma-Aldrich		AAAAGTTCCCCTGCTGATGATTTGT
Sequence-based reagent	Pten_flox-wt_geno_Rv	Sigma-Aldrich		TGTTTTTGACCAATTAAAGTAGGCTG
Sequence-based reagent	Cre_geno_Fw	Sigma-Aldrich		AGGTGTAGAGAAGGCACTTAGC
Sequence-based reagent	Cre_geno_Rv	Sigma-Aldrich		CTAATCGCCATCTTCCAGCAGG
Peptide, recombinant protein	EGF	Peprotech	315-09	10 ng/ml
Peptide, recombinant protein	hBMP5	Aviscera Bioscience	00013-01-100	50 ng/ml
Peptide, recombinant protein	hNOGGIN	RayBiotech	230-00704-100	180 ng/ml
Peptide, recombinant protein	Collagenase/hyaluronidase	Stemcell Technologies	. 07912
Peptide, recombinant protein	dispase II	Roche	4942078001	(5 U/ml)
Peptide, recombinant protein	DNase I	Roche	11284932001	0.1 mg/ml
Peptide, recombinant protein	matrigel	Corning	CACB354234
Peptide, recombinant protein	BPE	Life technologies	13028014	25 ug/ml
Peptide, recombinant protein	b27 SUPPLEMENT	Life technologies	17504044
Peptide, recombinant protein	collagenase A	Bioshop	COL004
Peptide, recombinant protein	fibronectin	Sigma-Aldrich	11051407001
Peptide, recombinant protein	collagen type I	Corning	354236
Peptide, recombinant protein	proteinase K	biobasic	PB0451
Peptide, recombinant protein	RNase A	Roche	10109169001
Commercial assay or kit	In Situ Cell Death Detection Kit	Roche	12156792910
Commercial assay or kit	P1 Primary Cell 4D-Nucleofector Kit	Bioscience	V4XP-1024
Commercial assay or kit	RNeasy micro kit	Qiagen	74004
Commercial assay or kit	Green-2-go mastermix	BioBasic	QPCR004
Commercial assay or kit	TruSeq Stranded Total RNA Sample Preparation Kit	Illumina
Chemical compound, drug	tamoxifen	Toronto Research Chemicals	T006000	3 mg
Chemical compound, drug	K02288	AdooQ Bioscience	A14311	10 uM
Chemical compound, drug	Y-27632	ApexBio	A3008	10 uM
Chemical compound, drug	4-hydroxy-tamoxifen	Toronto Research Chemicals	H954725	500 ng/ml
Chemical compound, drug	5α-dihydrotestosterone	steraloids	A2570-000	1nM
Chemical compound, drug	phosphatase inhibitor cocktails (Phostop)	Sigma	4906845001
Chemical compound, drug	Fixable Viability Dye eFluor 780	ebioscience	65-0865-18
Software, algorithm	Prism 6.0	GraphPad
Software, algorithm	ImageJ	Fiji
Software, algorithm	FlowJo	LLC
Software, algorithm	DIVA	BD Biosciences
Software, algorithm	L-calc	Stem cell technologies
Software, algorithm	matrix2png program	Pavlidis and Noble, 2003