Distinct microRNA expression profiles in prostate cancer stem/progenitor cells and tumor suppressive functions of let-7

Abstract

microRNAs (miRNAs) regulate cancer cells but their potential effects on cancer stem/progenitor cells are still being explored. In this study we used quantitative RT-PCR to define miRNA expression patterns in various stem/progenitor cell populations in prostate cancer (PCa), including CD44⁺, CD133⁺, integrin α2β1⁺ and side population cells. We identified distinct and common patterns in these different tumorigenic cell subsets. Multiple tumor suppressive miRNAs were downregulated coordinately in several PCa stem/progenitor cell populations, namely, miR-34a, let-7b, miR-106a and miR-141, whereas miR-301 and miR-452 were commonly overexpressed. let-7 overexpression inhibited PCa cell proliferation and clonal expansion in vitro and tumor regeneration in vivo. In addition, let-7 and miR-34a exerted differential inhibitory effects in PCa cells, with miR-34a inducing G1 phase cell-cycle arrest accompanied by cell senescence and let-7 inducing G2/M phase cell-cycle arrest without senescence. Taken together, our findings define distinct miRNA expression patterns that coordinately regulate the tumorigenicity of PCa cells.

Introduction

Most tumors contain a dynamic population of less differentiated and highly tumorigenic cells operationally defined as cancer stem cells (CSCs) or tumor initiating cells (1–10). CSCs may be phenotypically purified using surface markers. CD44 is one such marker widely used to enrich tumor-initiating cells, for example, in cancers of the breast (2), pancreas (5), head and neck (8), colon (9), and the prostate (6,7). Our previous work has shown that CD44⁺ cells from prostate cancer (PCa) cell cultures or PCa xenografts exhibit high proliferative and clonogenic potential in vitro. Moreover, using limiting dilution assays in NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice, we find that CD44⁺ PCa cells possess 6–30 times higher tumor-regenerating capacity compared to CD44⁻ cells (6,7). CD133 has similarly been utilized to enrich CSCs in brain (3), colon (10), and other cancers. Several surface marker-independent strategies have also been employed to enrich tumor-initiating cells (1). Side population (SP) assay is a flow cytometry-based method initially developed to enrich hematopoietic stem cells owing to their expression of high levels of drug-detoxifying surface transporter proteins such as ABCG2 and MDR1 that efficiently efflux the Hoechst dye 33342 (11). Using the SP technique, we have shown that the SP cells in LAPC9 xenografts, though representing only ~0.01% of the total tumor cell population, are >500 fold more tumorigenic than the isogenic non-SP cells (12).

With the preponderant evidence for CSCs and our increasing knowledge of CSC heterogeneity (1), it becomes apparent that we need to understand how tumorigenic cancer cells are regulated at the molecular level so that we can design CSC-specific therapeutics. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many biological processes by inhibiting the target mRNA translation or stability (13). Deregulation of miRNAs has been observed in a variety of human tumors (14,15). In PCa, several groups have performed miRNA expression profiling studies using either miRNA microarray (16–20) or whole-genome deep sequencing (21) in PCa cell lines, xenografts or patient samples. These studies, although reporting PCa-related miRNA alterations and shedding light on differential miRNA expression in PCa (relative to benign tissues), have all been performed in bulk tumor cells and thus fail to address alterations of miRNA expression and functions specifically in tumorigenic PCa cell subsets. We recently performed, for the first time, a miRNA expression profiling in 6 highly purified PCa stem/progenitor cell populations and reported that miR-34a, a p53 target, was under-expressed in all these populations (22). We further showed that miR-34a negatively regulated prostate CSC (PCSC) activity and inhibited PCa metastasis by directly repressing CD44 (22). Herein, we present detailed miRNA expression profiling procedures and results and report the miRNAs that are commonly and differentially expressed in PCa stem/progenitor cell populations. We further investigate the biological functions of two commonly altered miRNAs, i.e., let-7 and miR-301, in the context of regulating CSCs and PCa regeneration. Finally, using miR-34a as an example, we explore potential mechanisms that may be responsible for the differential miRNA expression in PCa stem/progenitor cells. Our results converge with the emerging theme that distinct miRNAs coordinately and distinctively regulate CSC properties (23).

Materials and Methods

Many basic experimental procedures have been described in our earlier publications (6,7,12,22,24–26). Some experimental procedures are described in Supplementary Methods. Primary human prostate tumors (HPCa) used in this study were presented in Supplementary Table S1.

Cells, xenografts, and animals

PPC-1, PC3, LNCaP and Du145 cells were obtained from ATCC and cultured in RPMI-1640 plus 7% heat-inactivated fetal bovine serum (FBS). Human xenograft prostate tumors, LAPC9 (bone metastasis; AR⁺ and PSA⁺), LAPC4 (lymph node metastasis; AR⁺ and PSA⁺), and Du145 (brain metastasis; AR⁻ and PSA⁻) were maintained in NOD/SCID mice. NOD/SCID mice were produced mostly from our own breeding colonies and purchased occasionally from the Jackson Laboratories (Bar Harbor) and maintained in standard conditions according to the Institutional guidelines. All animal experiments were approved by our institutional IACCUC. All these 6 PCa cell types were routinely checked to be free of mycoplasma contamination using the Agilent MycoSensor QPCR Assay kit (cat. #302107). Cell authentification by DNA fingerprinting is under way.

Transient transfection with oligonucleotides

PCa cells were transfected with 30 nM of miR-34a, let-7a, let-7b, or miR-301 mirVana mimics or non-targeting negative control miRNA (miR-NC) oligos (Ambion) using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions (22). MirVana mimics are synthetic double-stranded oligonucleotides (oligos) that mimic mature miRNAs. In some experiments, mirVana miRNA inhibitors, chemically modified antisense oligos against let-7b, miR-301, or miR-NC (Ambion) were introduced into PCa using the same conditions. After culturing for overnight to 48 h, transfected cells were harvested for in vitro and in vivo studies.

Lentiviral-mediated overexpression of let-7a

pLL3.7-let-7a and pLL3.7 control vector were kindly provided by Dr. J. Lieberman (Harvard University; 27). Lentiviruses were produced in 293FT packaging cells and titers determined for GFP using HT1080 cells (22). PCa cells were infected with the lentiviral supernatant (MOI 5–10) in the presence of 8 µg/ml polybrene and harvested 48–72 h post-infection for experiments.

Statistical analyses

In general, unpaired two-tailed Student’s t-test was used to compare differences in cell numbers, cumulative PDs, percentages of CD44⁺, % BrdU⁺ cells, % cell-cycle phases, cloning and sphere-formation efficiency, and tumor weights. Fisher’s Exact Test and χ² test were used to compare incidence and latency. In all these analyses, a P < 0.05 was considered statistically significant.

Results

miRNA expression profiling in purified PCa stem/progenitor cell populations

We first employed the quantitative RT-PCR (qPCR; 22) to determine the expression levels of 310 mature human miRNAs (Supplementary Table S2) in bulk PCa cells purified from 3 xenografts, i.e., LAPC9 (bone metastasis, AR⁺/PSA⁺), LAPC4 (lymph node metastasis, AR⁺/PSA⁺), and Du145 (brain metastasis, AR⁻/PSA⁻) (Supplementary Fig. S1, step I). We then chose 136 miRNAs (Supplementary Table S3) including the top 120 abundantly expressed miRNAs and 16 less abundant miRNAs of interest (including 2 miRNAs, i.e., miR-24 and miR-103, that were used as internal controls). We measured the levels of these 136 miRNAs in CD44⁺ (i.e., cells expressing high levels of CD44) and CD44⁻ cells purified from LAPC9, LAPC4 and Du145 tumors; CD133⁺ and CD133⁻ cells from LAPC4 tumor, and integrin α2β1⁺ and α2β1⁻ cells from Du145 tumor (Supplementary Fig. S1, step II). The LAPC9, LAPC4, and Du145 tumors contain ~20%, 0.1%, and 30% CD44^hi cells, respectively (6) whereas the LAPC4 tumors contain ~1% CD133⁺ cells. The CD44⁺ PCa cells are enriched in tumor- and metastasis-initiating cells (6,7) whereas CD133⁺(CD44⁺α2β1^hi) cells purified from primary PCa samples are highly clonogenic (28). In addition to these 5 (i.e., three CD44⁺, one CD133⁺, and one α2β1⁺) PCa cell populations, we also purified, from the LAPC9 tumor, the SP, which harbors great tumor-regenerative activity (12). Since the SP represents <0.1% of the total population in LAPC9 tumor (12), we manually curated 57 miRNAs (Supplementary Table S4) that could be reliably detected and compared their expression levels in the SP vs. non-SP cells (Supplementary Fig. S1, step III). Comparisons of 6 marker-positive and -negative PCa cell populations revealed interesting and informative differences in miRNA expression patterns.

Common under-expression of multiple tumor-suppressive miRNAs in CD44⁺ PCa cells

We first compared the expression levels of 134 miRNAs between the CD44⁺ and CD44⁻ populations and observed cell type-related differential miRNA expression patterns (Supplementary Fig. S2A–C; Supplementary Table S3). The CD44⁺ LAPC4 and LAPC9 cells had significantly more under-expressed than over-expressed miRNAs compared to the corresponding CD44⁻ cells whereas CD44⁺ and CD44⁻ Du145 cells had roughly similar numbers of over-expressed and under-expressed miRNAs (Supplementary Fig. S2). When we analyzed the miRNA expression patterns common to all 3 populations of CD44⁺ PCa cells, we found that 3 miRNAs, i.e., miR-452, miR-19a, and miR-301, were commonly over-expressed and 37 miRNAs were commonly under-expressed (Table 1; Supplementary Table S3). Among the 37 under-expressed miRNAs, miR-34a was most dramatically down-regulated, representing 2% of the level in CD44⁻ cells. We have recently shown that miR-34a acts as a critical negative regulator of PCSC properties by directly targeting CD44 (22). In addition to miR-34a, four let-7 members (let-7a, let-7b, let-7e and let-7f) were under-expressed in the 3 CD44⁺ populations (Table 1). Moreover, miR-141, a miR-200 family member, was also expressed at lower levels in CD44⁺ than in CD44⁻ PCa cells (Table 1). miR-34, let-7, and miR-200 families of miRNAs are well-established tumor-suppressive miRNAs (22,23,27,29,30).

Table 1.

miRNAs commonly over- or under-expressed in CD44⁺ PCa cells^#

Over-expressed		Under-expressed
miRNA	fold change	miRNA	fold change	miRNA	fold change
miR-452	832.77	miR-34a	0.02	miR-183	0.59
miR-19a	2.99	miR-199a*	0.04	miR-132	0.60
miR-301	1.84	miR-218	0.06	let-7e	0.62
		miR-422b	0.24	miR-340	0.62
		miR-422a	0.27	miR-30a-3p	0.64
		miR-378	0.27	miR-30a-5p	0.64
		miR-196a	0.28	miR-324-5p	0.64
		miR-10a	0.33	miR-365	0.66
		let-7b	0.35	miR-193b	0.67
		miR-214	0.39	miR-24	0.67
		miR-148a	0.41	miR-335	0.67
		miR-203	0.43	miR-191	0.68
		miR-181b	0.43	miR-92	0.70
		let-7a	0.44	miR-182	0.76
		miR-141	0.47	miR-99b	0.80
		miR-222	0.48	miR-30c	0.80
		miR-342	0.52	miR-106a	0.83
		let-7f	0.53	miR-19b	0.85
		miR-151	0.57

^#Presented are the miRNAs that are commonly over- or under-expressed in the purified CD44⁺ Du145, LAPC9, and LAPC4 cells compared to the corresponding CD44⁻ cells using miR-103 as the internal control. The fold changes represent the mean value of the miRNA in three xenograft models.

miR-199a*, which is downregulated in many cancers (in particular, hepatocellular carcinoma) and possesses tumor-suppressive functions by targeting oncogenic molecules such as c-MET, versican, PAK4, Brm, mTOR, and AKT (31–33), was expressed in CD44⁺ PCa cells at only ~4% levels of the CD44⁻ cells (Table 1). Strikingly, in other cancer cells, miR-199a* has been demonstrated to target CD44 leading to its deficiency in CD44⁺ cancer cells (32,33). Of interest, miR-214 is in a cluster with miR-199a* (~6 kb apart) within human dynamin-3 gene intron (DNM3os) and was co-downregulated with miR-199a* in CD44⁺ cells (Table 1). Similarly, miR-10a and miR-196a are embedded in the HoxB gene cluster and both were under-expressed in CD44⁺ PCa cells (Table 1). Several other clusters of miRNAs, including let-7e/miR-99b (19q13.33), miR-183/182 (7q31–34), miR106a/19b/92a (in the Chr-X mir-106a-363 cluster), and miR-193b/365 (16p13.12), were also coordinately down-regulated in the CD44⁺ PCa cells (Table 1). miR-193b targets multiple oncogenic molecules including uPA, cyclin D1, 14-3-3ζ, c-Kit, and Mcl-1 and is important for cellular differentiation (34). Many other miRNAs commonly under-expressed in CD44⁺ PCa cells (Table 1), including miR-218 (35), miR-148a (36), miR-181b (37), miR-203 (38), miR-183 (39), miR-24 (40), and miR-335 (41) all possess tumor/metastasis inhibitory functions.

Together, our profiling results indicate that multiple tumor-suppressive miRNAs are coordinately down-regulated in CD44⁺ PCa cells. We then employed the online database Diana mir-Path (42) to probe the potential signaling pathways that might be engaged by differentially expressed miRNAs. The software performs an enrichment analysis of multiple microRNA target genes against all known KEGG pathways. When we input the set of miRNAs commonly under-expressed in CD44⁺ cells, the top hits were TGFβ, Wnt, and MAPK signaling pathways (not shown).

The CD44⁺ PCa cells are generally less differentiated (e.g., expressing less AR) (6). Consistent with this notion, many of the miRNAs identified here to be under-expressed in the CD44⁺ LAPC9, LAPC4, and Du145 cells, including miR-34a, miR-141, let-7 members, miR-10a, miR-214, miR-203, miR-183, miR-365, miR-193b, miR-24, and miR-30c (Table 1) are generally depleted in (cancer) stem cells and preferentially expressed in differentiated progeny. In further support, several miRNAs under-expressed in CD44⁺ PCa cells, such as miR-148a (43) and miR-141 (44), have been shown to be androgen-responsive.

Distinctive and common miRNA expression profiles in PCa stem/progenitor cell populations

We then analyzed the expression levels of 134 miRNAs in LAPC4 CD133⁺ and Du145 α2β1⁺ cells, and 57 miRNAs in LAPC9 SP cells in comparison to their corresponding marker-negative populations and we observed miRNA expression patterns unique to each tumor cell population (Fig.1; Supplementary Table S3–S4). Interesting, the top over-expressed miRNA in CD133⁺ LAPC4 cells was miR-21, one of the best characterized oncomiRs widely overexpressed in human cancers (45). Among the top 10 down-regulated miRNAs were many that were also under-expressed in CD44⁺ PCa cells and several tumor-suppressive miRNAs including miR-133a, miR-126, miR-15a and miR-200a (Fig. 1A). In general, the magnitude of downregulation (i.e., to ~10⁻⁶) was much more pronounced than that of upregulation (up to ~10² for most) although surprisingly, there were more miRNAs overexpressed than under-expressed in CD133⁺ LAPC4 cells (Fig. 1A). When we compared the 134 miRNA expression in CD133⁺ vs. CD44⁺ LAPC4 cells, we observed 25 commonly overexpressed and 29 commonly under-expressed miRNAs (Supplementary Fig. S3A).

Figure 1. miRNA expression profiles in CD133⁺, α2β1⁺, and side population (SP) PCa cells.

A–C. Presented are the miRNA expression levels in the marker-positive populations including CD133⁺ from LAPC4 (A), side population from LAPC9 (B), and α2β1⁺ from Du145 (C), relative to the corresponding marker-negative populations. The top 10 over- and under-expressed miRNAs are listed on the right.

In contrast to CD133⁺ PCa cells, there were significantly more miRNAs under-expressed than overexpressed in LAPC9 SP cells when compared to the isogenic non-SP cells and, again, the levels of downregulation were higher than those of upregulation (Fig. 1B). The top overexpressed miRNA was miR-451, which was recently shown to regulate the self-renewal and tumorigenicity of colorectal CSCs (46). Among the top 10 under-expressed miRNAs in SP were miR-15a/15b and several oncosuppressive miRNAs downregulated in CD44⁺ PCa cells. We observed 6 commonly overexpressed and 31 commonly under-expressed miRNAs in SP vs. CD44⁺ LAPC9 cells (Supplementary Fig. S3B). Finally, roughly similar numbers of up- and down-regulated miRNAs were observed in α2β1⁺ and α2β1⁻ Du145 cells (Fig. 1C). We observed 44 commonly overexpressed and 22 commonly under-expressed miRNAs in α2β1⁺ vs. CD44⁺ Du145 cells (Supplementary Fig. S3C). Surprisingly, among the top 10 upregulated miRNAs were miR-30a-5p, let-7a, and miR-196a (Fig. 1C), which were commonly under-expressed in CD44⁺ PCa cells (Table 1). These observations are consistent with our earlier conclusions that the α2β1⁺ PCa cell population overlaps with but is also distinct from the CD44⁺ population (7).

Subsequently, we tried to identify commonly changed miRNAs. We first compared the common CD44 profiles (Table 1) with the profiles generated from CD133⁺ or α2β1⁺ populations (Supplementary Table S2–S3), and uncovered the miRNAs that were commonly over- or under-expressed in the 4 (i.e., 3 CD44⁺ together with CD133⁺ or α2β1⁺) cell populations (Table 2). When we combined 5 populations (i.e., 3 CD44⁺ together with CD133⁺ and α2β1⁺), only 4 miRNAs, i.e., let-7b, miR-106a, miR-141 and miR-34a, were commonly under-expressed and 2 miRNAs, i.e., miR-301 and miR-452, were commonly over-expressed (Fig. 2A; Table 2). When we further included the expression profile from the LAPC9 SP, only one miRNA, i.e., miR-34a, was commonly under-expressed and one miRNA, miR-452, was commonly over-expressed in all 6 PCa cell populations (Table 2; Supplementary Fig. S4A).

Table 2.

Commonly over- and under-expressed miRNAs in tumorigenic PCa cell populations

4 populations (a)		4 populations (b)		5 populations (c)		6 populations (d)
Over- expressed	Under- expressed	Over- expressed	Under- expressed	Over- expressed	Under- expressed	Over- expressed	Under- expressed
miR-19a	miR-34a	miR-301	miR-34a	miR-301	miR-34a	miR-452	miR-34a
miR-301	let-7b	miR-452	let-7b	miR-452	let-7b
miR-452	miR-106a		miR-106a		miR-106a
	miR-141		miR-141		miR-141
	let-7f		let-7e
	miR-335		miR-183
	miR-340		miR-203
	miR-365		miR-218
	miR-92		miR-342
			miR-378
			miR-422a
			miR-422b

^a.These 4 populations refer to the 3 CD44⁺ populations from LAPC9, LAPC4 and Du145, respectively, plus the CD133⁺ population from LAPC4.

^b.These 4 populations refer to the 3 CD44+ populations plus the α2β1⁺ population from Du145.

^c.These 5 populations refer to 3 CD44⁺ populations from LAPC9, LAPC4 and Du145 plus the CD133⁺ population from LAPC4 and the α2β1⁺ population from Du145.

^d.The 6 populations include the 5 populations in c plus the LAPC9 side population (SP).

Figure 2. Commonly under- and over-expressed miRNAs in tumorigenic populations of PCa cells and validation in CD44⁺ HPCa cells.

A. Four commonly under-expressed miRNAs (left) and two commonly over-expressed miRNAs (right) in 5 marker-positive PCa cell populations (see bar legend). Shown are the miRNA expression levels (%) in the marker-positive populations relative to those in the corresponding marker-negative populations.

B–C. Validation of let-7b (B) and miR-301 (C) expression in purified CD44⁺ HPCa cells. Shown are the mean value of the relative expression in CD44⁺ over CD44 HPCa cells. Note that the actual miR-301 expression level in CD44⁺ HPCa79T (the last bar in C) was 96848.63% relative to the corresponding CD44 HPCa79T cells.

Validation of commonly changed miRNAs in patient tumor (HPCa) derived CD44⁺ cells

The preceding miRNA library expression profiling was conducted in cells purified from three xenograft models. To validate the miRNA expression data, we purified CD44⁺ and CD44⁻ PCa cells from 21 primary HPCa samples (Supplementary Table 1), and measured the levels of 4 commonly under-expressed (miR-34a, let-7b, miR-141, and miR-106a) and 2 commonly overexpressed (miR-301 and miR-452) miRNAs. This strategy has an additional advantage of establishing the potential clinical relevance. We previously verified miR-34a under-expression in all HPCa-purified CD44⁺ PCa cells (22). let-7b also showed under-expression in the majority (18 out the 21) of samples in the CD44⁺ HPCa cells (Fig. 2B). Likewise, miR-141 was detected at much lower levels in CD44⁺ than CD44⁻ cells derived from most HPCa samples (data not shown). In contrast, miR-106a was under-expressed in 3 of the 5 xenograft-derived populations (Supplementary Fig. S4B) and in only ~50% of 21 HPCa-derived CD44⁺ PCa cells (Supplementary Fig. S4C). With the 2 commonly overexpressed miRNAs, we detected an over-representation of miR-301 in the CD44⁺ cells in 18 of the 21 HPCa samples (Fig. 2C). Unexpectedly, although miR-452 was dramatically upregulated in 4 of the 5 xenograft populations (Supplementary Fig. S4D), it was downregulated in most CD44⁺ HPCa cells (Supplementary Fig. S4E). Altogether, of the 6 miRNAs commonly changed in the 5 PCa cell populations, we could corroborate the under-expression of miR-34a, let-7b, and miR-141 and overexpression of miR-301 (i.e., 4/6 or 67%) using primary tumor-derived CD44⁺ HPCa cells.

let-7 inhibits clonal and sphere formation in PCa cells: Differential effects from miR-34a

To investigate the biological functions of commonly and differentially expressed miRNAs, we first focused on two under-expressed miRNAs, i.e., miR-34a and let-7, mainly because both had been demonstrated to possess strong tumor-suppressive functions in other systems (27,29,30). Our earlier studies demonstrated that miR-34a functioned as a negative regulator of PCSCs and PCa metastasis (22). Herein we focused on let-7 since four let-7 miRNA family members were under-expressed in CD44⁺ cells (Table 1) and let-7b was commonly under-expressed in 5 PCa cell populations (Fig. 2A) as well as in CD44⁺ HPCa cells (Fig. 2B). Over-expression of let-7b in Du145 cells by transfection of a let-7b mimicking oligonucleotide reduced cell number (Fig. 3A) due to inhibition of proliferation as assessed by BrdU incorporation assays (Fig. 3B). In addition, let-7b oligos, when compared to the negative control (NC) oligos that contain a scrambled sequence, inhibited the establishment of Du145 holoclones (Fig. 3C–E) and spheres (Fig. 3F). PCa cell holoclones contain self-renewing tumor-initiating cells (24) and PCa cell spheres formed under anchorage-independent conditions harbor tumor-initiating cells (6,12,25). Finally, when we infected Du145 cells with a lentivirus (i.e., pLL3.7-let7a; 27) that encodes let-7a (which recognizes the same seed sequence as let-7b), both clonal development (Supplementary Fig. S5A) and sphere formation (Supplementary Fig. S5B–C) were inhibited. We observed similar inhibitory effects of let-7b oligos in another PCa cell type, PPC-1 (Fig. 3G; Supplementary Fig. 6A–C). It should be noted that all miRNA mimicking oligos used in our previous (22) and present studies are mature miRNAs, which mimic the dicer cleavage product loaded into the RISC in the cytoplasm (22).

Figure 3. Biological effects of let-7b on PCa cells in vitro.

A–B. let-7b inhibits Du145 cell proliferation. A. 1000 cells transfected with NC or let-7b oligos (30 nM) were plated in 6-well plate on day 0 and cells were trypsinized and counted on days indicated. Average cell numbers were plotted. Presented in B is the mean % of BrdU-positive cells counted from a total of 800–1,000 cells.

C–E. Clonal assays in Du145 cells. Cells transfected with the indicated oligos (30 nM; 24 h) were plated in 6-well plate at clonal density. The plating cell numbers and the days when holoclones (a representative picture shown in the insert, C) were enumerated are indicated.

F. Sphere formation assays in Du145 cells. Cells were transfected as above and 3000 cells mixed with Matrigel were plated in 6-well plate for sphere formation assay. Spheres were counted in ~2 weeks. A representative sphere is shown in the insert.

G. Over-expression of let-7b inhibits PPC-1 clonal growth. Cells (100) transfected with the indicated oligos (30 nM; 48 h) were plated in triplicate in 6-well culture plates. The three types of clones were enumerated 10 days after plating. *P < 0.05 when compared with the NC group. Let-7b (and miR-34a) also reduced the total number of clones (P<0.05).

H–I. miR-34a induces PPC-1 cell G1 arrest whereas let-7b causes G2/M phases arrest. Cells were transfected with the oligos (30 nM; 48 h) followed by cell-cycle analysis by flow. Shown in H are representative histograms and in I is the quantification (n = 4).

J. Senescence-associated β-gal staining. Shown are the SA-βgal⁺ cells. All assays were done in triplicate.

Overall, let-7b mimicking oligos demonstrated similar inhibitory effects to miR-34a overexpression on PCa cell holoclones and spheres (Fig. 3C–G; Supplementary Fig. S6B–C). However, when we analyzed cell-cycle profiles in PPC-1 cells treated with miR-34a or let-7b oligos, we observed that miR-34a caused G1 cell-cycle arrest whereas let-7b led to prominent G2/M phase arrest (Fig. 3H–I). Fully consistent with the differential effects between miR-34a and let-7b on cell cycle, miR-34a overexpression induced significantly increased cell senescence assessed by staining of PCa cells for senescence-associated β-gal (SA-βgal) activity (Fig. 3J; Supplementary Fig. S6D). It is well documented that G1 cell-cycle arrest generally precedes cell senescence. In contrast, let-7b oligos, which did not cause G1 arrest, did not induce PPC-1 cell senescence (Fig. 3J; Supplementary Fig. S6D). These results altogether suggest that let-7b and miR-34a exert differential mechanisms in PCa cells with respect to their effects on cell cycle and senescence.

let-7 inhibits prostate tumor regeneration: Evidence for fast turnover of let-7 in PCa cells

Next, we investigated the let-7 effects on tumor regeneration. We first performed the ‘positive’ control experiments by subcutaneously (s.c) implanting A549 lung cancer cells that had been transfected with the let-7b or NC oligos in NOD/SCID mice. As reported earlier by others (30), let-7b overexpression suppressed A549 tumor development (Supplementary Fig. S7A). Surprisingly, in multiple similar tumor experiments performed in Du145 (Supplementary Fig. S7B–C) or LAPC9 (Fig. 4A; Supplementary Fig. S7D) cells, let-7b oligos did not manifest obvious tumor-suppressive effects whether cells were implanted s.c or in the dorsal prostate (DP; Supplementary Fig. S7B–C). Similar to let-7b oligos, let-7a oligos also did not inhibit LAPC9 tumor regeneration although miR-34a oligos significantly retarded tumor growth (Fig. 4A). These surprising results suggested that 1) let-7 miRNAs might exert differential effects on lung (A549) vs. prostate (Du145 and LAPC9) cancer cells; 2) transfected let-7 oligos might be turned over faster in PCa cells compared to lung cancer cells; 3) let-7 and miR-34a might exert divergent regulatory roles in PCa cells; and/or 4) let-7 oligos might become degraded or turned over faster than miR-34a oligos in PCa cells.

Figure 4. Effects of let-7 on prostate tumor development in vivo.

A. Over-expression of let-7b or let-7a in LAPC9 cells by oligo transfection did not inhibit tumor regeneration. Shown are tumor images harvested at 44 days post implantation. The mean tumor weight and incidence are indicated on the right.

B–C. RNA levels of let-7a/b or miR-34a in freshly transfected (30 nM; 48 h) Du145 (B), or LAPC9 and A549 (C) cells measured by qRT-PCR. Shown are the expression levels (fold) relative to the corresponding NC control. In C, the differences between miR-34 and let-7a or let-7b mRNA levels in LAPC9 cells were statistically significant (P < 0.05 for both; based on comparisons of the actual ddCt values). Also, the let-7b mRNA levels in A549 cells were significantly higher than those in LAPC9 (P = 0.019; ddCt) or in Du145 (P = 0.049; ddCt) cells.

D. RNA levels of let-7a/b or miR-34a in endpoint LAPC9 or A549 tumors measured by qRT-PCR. Shown are the expression levels (fold) relative to the corresponding NC control. These endpoint tumors were derived from the corresponding transfected cells shown in A or C.

E–F. Over-expression of let-7a by lentiviral-mediated transduction significantly inhibited LAPC9 tumor development. In E, tumor sizes were measured by a caliber on the days indicated. In F, tumor images, average tumor weight and incidence (parenthesis) were presented.

G. Over-expression of let-7a inhibited tumor development from CD44⁺ Du145 cells.

H–I. Validation of let-7 downstream targets by qPCR (H) and Western blotting (I) analysis. In H, Du145 cells were transfected with let-7b or NC oligos (30 nM; 48 h), and cells were harvested for qPCR analysis of K-Ras, c-Myc and Bcl-2 mRNAs. In I, Du145 cells were transfected with the oligos (conc. and time indicated). Cells were lysed and 50 µg of whole cell lysate was loaded in each lane for the molecules indicated. Relative densitometric values were given below.

To start addressing these possibilities, we first measured let-7a/b and miR-34a levels in both freshly transfected cells and endpoint tumors (Fig. 4B–D). Du145 (Fig. 4B) and LAPC9 (Fig. 4C) cells transfected with let-7 oligos had several hundred fold higher levels of let-7 than the same cells transfected with NC oligos at 48 h. Unexpectedly, however, A549 cells transfected with the same amount (i.e., 30 nM) of let-7b possessed much higher levels of intracellular let-7b than either Du145 or LAPC9 cells (Fig. 4B–C). More surprisingly, at 48 h after transfection of the same amount of miR-34a or let-7b (30 nm for each), LAPC9 cells retained significantly higher levels of miR-34a than let-7b (Fig. 4C). As expected, the endpoint tumors all expressed similarly low levels of let-7a/b or miR-34a (Fig. 4D). These results suggest that transfected let-7 oligos, in contrast to miR-34a oligos, were rapidly degraded in PCa cells, in contrast to A549 cells. Consistent with this suggestion, when we infected LAPC9 cells with pLL3.7-let-7a, the continuously delivered let-7a significantly slowed tumor growth (Fig. 4E) and inhibited tumor regeneration (Fig. 4F). Impressively, pLL3.7-let-7a also inhibited tumor development of the purified CD44⁺ Du145 cells (Fig. 4G).

The let-7 family miRNAs repress many oncogenic molecules including Ras, c-Myc, HMG, and Bcl-2 (27,29,30). We observed that PCa cells freshly transfected with the let-7b oligos exhibited significantly reduced c-Myc and K-Ras, both at the mRNA (Fig. 4H) and protein (Fig. 4I) levels. Luciferase reporter assays confirmed K-Ras as a direct let-7 downstream target (Supplementary Fig. S8). In contrast, the Bcl-2 mRNA and protein levels were not affected by let-7b (Fig. 4H–I).

miR-301 exerted differential biological effects on different PCa cells

We also probed for the biological functions of one commonly overexpressed miRNA, i.e., miR-301 (Supplementary Fig. S9–S10). Unexpectedly, enforced miR-301 expression via oligo transfection in purified CD44⁻ Du145 cells (Supplementary Fig. S9A) or knocking down endogenous miR-301 in CD44⁺ Du145 cells (Supplementary Fig. S9B) did not significantly affect sphere formation. Similar negative results were obtained in holoclone assays (Supplementary Fig. S9C–D). miR-301 overexpression and knockdown were verified by qPCR (Supplementary Fig. S9E). Manipulation of miR-301 levels also did not affect the tumor regeneration of CD44⁺/CD44⁻ Du145 cells (Supplementary Fig. S9F–I). Similarly, anti-miR-301 oligos did not alter the clonal and tumorigenic properties of PC3 cells (Supplementary Fig. S10A–C). In contrast, enforced miR-301 expression promoted whereas anti-miR-301 reduced the clonal and sphere-forming capacities of xenograft-derived LAPC9 cells (Supplementary Fig. S10D–E).

How miRNAs might be under-expressed in PCa stem/progenitor cells?

How tumor-suppressive miRNAs such as miR-34a and let-7 might be under-expressed in tumorigenic subpopulations is an interesting question. We attempted to address this question by focusing on miR-34a, whose expression is regulated in both p53-dependent and p53-independent mechanisms (47). The miR-34a levels in the 4 PCa cell types with null or mutant p53 were significantly lower than those in the 6 prostate (cancer) cell types with wild-type (wt) p53 (22). To explore whether the lower levels of miR-34a in tumorigenic PCa cells might be related to lower p53 expression/activity, we treated p53-wt LNCaP cells with paclitaxel and three DNA-damaging agents, i.e., doxorubicin, etoposide, and γ-irradiation (X-ray). p53 was activated by etoposide and X-ray, as evidenced by both p53 protein accumulation (Fig. 5A) and increased protein and mRNA levels of p21 (Fig. 5A–B), a p53 transcriptional target. When miR-34a (1p36.22) and miR-34b/c (11q23.1) levels were measured in treated LNCaP cells, we observed that miR-34a levels did not significantly change except a slight increase at 48 h (Fig. 5C). In contrast, both etoposide and X-ray increased miR-34b and miR-34c levels by several fold (Fig. 5C). These observations suggest that under-expression of miR-34a in CD44⁺ PCa cells might not be related to p53 expression or activity. In support, the miR-34a mRNA levels in the CD44⁺ cells freshly purified from 14 primary HPCa cells did not correlate with p53 (Fig. S11A–B) or p21 (not shown) mRNA levels. Previous studies suggest that c-Myc may positively regulate miR-34a (48). However, the miR-34a mRNA levels in CD44⁺ HPCa cells also did not correlate with c-Myc mRNA (Supplementary Fig. S11C).

Figure 5. p53 activation in LNCaP cells differentially affects miR-34 family members.

A. LNCaP cells were treated with paclitaxel (Taxol, 25 nM), doxorubicin (Dox, 10 ng/ml), etoposide (Etop, 50 nM), or γ-irradiation (X-ray, 10 Gy) for the time intervals indicated. Whole cell lysate (50 µg/lane) was used in Western blotting for p53, p21, or GAPDH (loading control).

B. Verification by qRT-PCR of upregulation of p21 mRNA in treated LNCaP cells.

C. p53 activation in LNCaP cells preferentially induces miR-34b and miR-34c over miR-34a. Among the 4 treatments, only γ-irradiation slightly increased miR-34a levels (left panel). In contrast, both etoposide and X-ray upregulated miR-34b expression (middle) whereas all 4 treatments increased miR-34c expression (right panel).

Discussion

For the first time, we have profiled the miRNA expression patterns in purified subpopulations of PCa cells that possess stem/progenitor cell properties. Among the CD44⁺, SP, CD133⁺, and α2β1⁺ cells studied, the CD44⁺ PCa cells are best characterized and have been consistently shown to enrich for tumor-initiating and pro-metastatic cells (6,7,22,25). The SP is also enriched in tumorigenic cells although it is more rare (<0.1%) and detectable only in LAPC9 cells (12,25). The CD133⁺ PCa cells are clonogenic and may also harbor CSCs (28). In contrast, the α2β1⁺ PCa cells are proliferative progenitors that do not enrich for CSCs (7). Our current miRNA profiling substantiates the heterogeneous nature of PCa stem/progenitor cells as CD44⁺, SP, CD133⁺, and α2β1⁺ cell populations exhibit overall distinct miRNA expression profiles. This is perhaps best illustrated by comparing CD44⁺ and CD133⁺ populations – the CD44⁺ PCa cells predominantly downregulate whereas the CD133⁺ LAPC4 cells significantly upregulate multiple miRNAs. Some of the under-expressed miRNAs such as miR-34a and let-7 are also down-regulated in prostate tumors in comparison to benign tissues (16–20).

On the other hand, different PCa stem/progenitor cells also commonly over- and under-express certain miRNAs. One of the most striking observations is that the 3 populations of CD44⁺ PCa cells commonly and coordinately down-regulate 37 miRNAs, many of which exist in genomic clusters and most of which possess tumor-suppressive functions. This observation is remarkably similar to the findings that multiple tumor-suppressive miRNAs are coordinately ‘depleted’ in other CSCs, e.g., the under-expression of let-7 family, miR-200 family, and miR-30 in breast CSCs and of miR-34a, miR-128, miR-451, and others in glioblastoma stem cells (reviewed in 23). More remarkably, Shimono et al also reported 37 miRNAs differentially expressed in the CD44⁺CD24^−/lo breast CSCs including the downregulation of 3 clusters, miR-200c/141, miR-200b/200a/429, and miR-183/96/182 (39), which are also downregulated in CD44⁺/α2β1⁺ PCa cells. Because these CSC-depleted miRNAs generally target potent oncogenic molecules involved in regulating cell cycle and proliferation (e.g., E2F, HMGA2, Ras, cyclins, CDKs), cell survival (e.g., Bcl-2, Mcl-1, Bcl-XL), self-renewal (e.g., Bmi, Notch, Myc), and cell migration/invasion (e.g., CD44, c-MET, ZEB) (23), it is conceivable that lack of these miRNAs would confer many stem cell properties. Many of these tumor-suppressive miRNAs are also deficient in embryonic and adult stem cells and preferentially expressed in differentiated progeny (23). Consequently, their lower expression in CD44⁺ PCa cells further supports the stem-like features of these cells and is consistent with earlier observations that the CD44⁺ PCa cells are less differentiated expressing little AR (6). In this regard, it is interesting that at least two androgen-responsive miRNAs, i.e., miR-148a (43) and miR-141 (44) are under-expressed in the CD44⁺ PCa cells. In contrast, another androgen-regulated miRNA, miR-21 (49), is the most highly expressed miRNA in CD133⁺ LAPC4 cells, emphasizing the difference between these two populations of PCa cells.

More miRNAs are down-regulated than up-regulated in the 3 CD44⁺ PCa cell populations in common with either CD133⁺ or α2β1⁺ cells (Table 2). When these 5 populations are combined for analysis, 4 miRNAs (i.e., miR-34a, let-7b, miR-106a, and miR-141) are commonly down-regulated and 2 miRNAs (i.e., miR-301 and miR-452) are commonly upregulated. Using the CD44⁺ HPCa cells freshly purified from patient tumors, we have confirmed the differential expression of 4 of the 6 (i.e., miR-34a, let-7b, miR-141, and miR-301) miRNAs in marker-positive vs. marker-negative cells. It is presently unclear why the under-expression of miR-106a and over-expression of miR-452 observed in 3 xenograft PCa cells are not borne out in CD44⁺ HPCa cells.

To establish whether the miRNAs identified in our miRNA library screening are functionally relevant, we have by far thoroughly studied two commonly under-expressed (i.e., miR-34a and let-7b) and one commonly over-expressed (i.e., miR-301) miRNAs. Our earlier studies have uncovered a powerful role of miR-34a in restricting PCSC activity and PCa regeneration/metastasis via repressing CD44 itself (22). In the present study, we report similar PCa-suppressive functions of let-7b/a. Our observations are in line with the widely recognized tumor-inhibitory effects of let-7a/b (27,29,30) and suggest that like miR-34a, the under-expressed let-7 normally functions to inhibit certain PCSC properties. An intriguing finding is that in PCa cells, the transfected mature let-7a/b oligos seem to be degraded much more rapidly than miR-34a oligos, explaining why the former do not manifest obvious tumor-inhibitory effects. In fact, even PCa cells infected with the pLL3.7-let-7a lentiviral vectors, which do manifest PCa-inhibitory effects, keep low steady-state levels of let-7a (Liu et al., unpublished observations). Coupled with the observations in A549 lung cancer cells, our work suggests that in PCa, let-7 miRNAs have a faster turnover rate than other miRNAs such as miR-34a. Future work will further explore this potentially interesting phenomenon. Another interesting finding is that let-7 and miR-34a possess mechanistic differences in suppressing PCa stem/progenitor cells: miR-34a induces G1 cell-cycle arrest followed by cell senescence whereas let-7 causes a prominent G2/M phase arrest without inducing senescence. Furthermore, miR-34a, but not let-7, induces apoptosis in some PCa cells (22). In support, let-7 overexpression does not affect the prosurvival molecule Bcl-2 (Fig. 4I).

In contrast to consistent PCa-inhibitory effects of miR-34a and let-7, miR-301, which is commonly over-expressed in PCa stem/progenitor cells and recently shown to promote breast cancer cell proliferation and invasion (50), seems to exhibit cell type-dependent effects. Although manipulation of miR-301 levels does not affect Du145 and PC3 cells, its overexpression promotes whereas its knockdown inhibits the clonogenic properties of LAPC9 cells.

It will be of general interest to understand how certain miRNAs are differentially expressed in CSCs vs. non-CSCs. Since the two populations are isogenic, it stands to reason that the differential expression results from epigenetic events rather than genetic mutations. Indeed, many tumor-suppressive miRNAs (e.g., miR-34a) are downregulated in cancer due to promoter hypermethylation or aberrant histone modifications. When we treated CD44⁺ PCa cells with 5-aza-deoxycytidine and/or trichostatin A (an inhibitor of histone deacetylase), we did not observe any significant increase in miR-34 (Liu et al., unpublished observations). The miR-34a levels also do not correlate with the two known upstream transcriptional regulators, i.e., p53 and c-Myc. In fact, even in p53-wt bulk LNCaP cells, p53 activation does not consistently result in significant upregulation of miR-34a. Altogether, these observations argue that some other mechanisms might be operating to dampen miR-34a expression in PCSC-enriched cells.

In summary, we have successfully performed a miRNA expression profiling study in several PCa stem/progenitor cell populations, which has revealed both distinctively and commonly expressed miRNAs in tumorigenic PCa cells. While shedding important light on how PCSCs may be regulated by miRNAs, our results converge with the emerging theme that distinct miRNAs both distinctively and coordinately regulate CSC properties (23). Finally, our study establishes that tumor-suppressive miRNAs identified herein, such as miR-34a and let-7b/a, may represent novel therapeutics to specifically target CSCs and can be used in replacement therapy regimens.