Progesterone receptor A promotes invasiveness and metastasis of luminal breast cancer by suppressing regulation of critical microRNAs by estrogen

Identification of microRNAs that are uniquely regulated by low-dose progesterone through PR-A

Progesterone bound to PR-A could impinge on regulation of invasiveness by E₂/ER in a manner that is mediated by one or more miRNAs in the following ways (depicted as schematics in Fig. 1, A and B). First, progesterone/PR-A could either activate or repress miRNA(s) (independent of estrogen), and this would then result in rescue of invasiveness from estrogen repression (Fig. 1A). On the other hand, E₂/ER could either activate or repress miRNA(s), resulting in inhibition of invasiveness; in this case, progesterone/PR-A could suppress regulation of the miRNAs by E₂, resulting in rescue of invasiveness from estrogen repression (Fig. 1B). To identify these putative miRNAs, we used the following cell line models: T47D-A (ER+/PR-A+/PR-B-null), T47D-B (ER+/PR-A-null/PR-B+), parental T47D (ER+/PR-A+/PR-B+), BT474 (ER+/PR-A+/PR-B+), and ZR-75–1 (ER+/PR-A+/PR-B+).

To identify candidate miRNAs in the putative pathways illustrated in Fig. 1A, we first undertook Affymetrix miRNA profiling to screen for miRNAs activated or repressed by PR-A but not PR-B at a low dose (1 nm) of R5020. This was accomplished by using Affymetrix miRNA profiling of miRNA changes in hormone-depleted T47D-A versus T47D-B cells treated with R5020 followed by data validation by quantitative RT-PCR. We found five miRNAs that were activated by low-dose progestin and PR-A (i.e. in T47D-A cells) but not by PR-B (i.e. in T47D-B cells). They are miR-6805-5p, miR-584-5p, miR-1228-5p, miR-501-5p, and miR-668-5p. These five miRNAs were also strongly up-regulated in other PR-A–positive cell lines, including parental T47D cells, BT474 cells, and ZR-75-1 cells (Fig. 1C; values in Table S1). The same Affymetrix miRNA analysis did not reveal any miRNAs that were repressed via PR-A alone.

In contrast to progestin-regulated miRNAs, there are considerable literature data on miRNAs regulated by E₂ acting through ER (Table S2). All of these miRNAs that were either activated or repressed by E₂ were tested by real-time RT-PCR analysis to identify miRNAs whose regulation by E₂ was prevented by R5020 in T47D-A cells (i.e. via PR-A) but not in T47D-B cells (i.e. via PR-B). In this manner, 10 miRNAs were identified; five among them (miR-17-5p, miR-20a-5p, miR-92a-3p, miR-106a-5p, and miR-106b-5p) were activated by E₂, and five (miR-26b-5p, miR-27a-5p, miR-27b-5p, miR-200c-5p, and Let7a-5p) were repressed by E₂ in the absence of progestin/PR-A. The counterregulation of these E₂-regulated miRNAs by progestin/PR-A was also confirmed in the other PR-A–positive cell lines, including parental T47D cells, BT474 cells, and ZR-75–1 cells (Fig. 1, D and E; values in Tables S3 and S4).

Finally, to ensure that higher doses of progestin did not enable PR-B to mimic regulation of the 15 miRNAs by PR-A, T47D-B cells were treated with vehicle, 1 nm E_2, and 1 nm E₂ plus R5020 (1–50 nm). There was no progestin dose–dependent miRNA activation or opposition to E₂ regulation by PR-B (Fig. S1).

Regulation of microRNAs by PR-A is sensitized to lower doses of progestin by overexpression of PR-A to a level observed in tumors

We have previously reported that progestin-induced rescue of invasiveness from estrogen repression is dependent exclusively on PR-A and is unaffected by co-expression of PR-B. We have also reported that, compared with T47D cells, in the isogenic recombinant T47D-PR-A++ cells that stably express a 3.6-fold higher level of PR-A (Fig. 2E and Fig. S2), lower doses of progesterone can rescue invasiveness from estrogen repression (13). To ensure the clinical relevance of the PR-A expression level in the T47D-PR-A++ model, we examined primary luminal breast tumor specimens by Western blotting. As the pathologists' definition of hormone receptor positivity of breast tumors may include tumors in which as few as 10% and often even 1% of the tumor cells show receptor expression, and as the distribution of the receptor-positive cells within a tumor could be heterogeneous, we tested lysates from specimens obtained from 53 patient tumors to ensure an adequate number of samples in which we could detect PR expression by Western blotting. As the tumors could also have variable ratios of epithelial to stromal tissue, we also probed the blots for the epithelial marker E-cadherin. Clearly, the tumor specimens frequently showed levels of PR-A expression that were comparable with or higher than that in T47D-PR-A++ cells (Fig. 2A), and the expression of PR-A was also frequently higher than that of PR-B in the tumors (Fig. 2A). These data confirm the clinical relevance of the isogenic T47DA++ cell model.

In T47DA++ cells, every one of the above 15 miRNAs was optimally regulated at or below 0.2 nm R5020, whereas the parental T47D cells required 0.5–1 nm R5020 for the same level of regulation (increase or decrease in the absence or presence of 1 nm E₂) (Fig. 2, B–D; values in Table S5). This level of hypersensitization to progestin of miRNA regulation in T47D-PR-A++ cells corresponds to the previously reported progestin dose dependence of invasiveness of T47D-PR-A++ cells versus parental T47D cells (13).

miR-92a-3p and miR-26b-5p are functionally linked to hormonal control of invasiveness

We tested the ability of E₂ to repress invasiveness or of R5020 to rescue invasiveness following inhibition of each of the above 15 miRNAs in T47D and BT474 cells. Inhibition was confirmed by reduction in target miRNA 72 h post-transfection, although residual miRNA-inhibitor duplexes may also be detected.

We first inhibited each of the progestin/PR-A–activated (but not E₂-regulated) miRNAs: miR-6805–5p, miR-584–5p, miR-1228-5p, miR-501-5p, and miR-668-5p (Fig. 3A). Invasiveness was unaffected in all cases in the presence of E₂ and R5020 (Fig. 3, B and C), indicating that these miRNAs do not have a role in hormonal cross-talk between progestin/PR-A and E₂/ER that influences invasiveness.

Next, we inhibited the five miRNAs whose activation by E₂ was blocked by progestin (miR-17-5p, miR-20a-5p, miR-92a-3p, miR-106a-5p, and miR-106b-5p) (Fig. 3D). Inhibition of miR-92a-3p alone abrogated the ability of E₂ to repress invasiveness in both T47D and BT474 cells (Fig. 3, E and F). Therefore, the ability of progestin/PR-A to block activation of miR-92a-3p by E₂ must be critical for the functional cross-talk of progesterone via PR-A.

Finally, we inhibited the five miRNAs whose repression by E₂ was blocked by progestin (miR-26b-5p, miR-27a-5p, miR-27b-5p, miR-200c-5p, and Let7a-5p) (Fig. 3G). Inhibition of miR-26b-5p alone abrogated the ability of progestin to rescue invasiveness from E₂ repression in both T47D and BT474 cells (Fig. 3, H and I). Therefore, the ability of progestin/PR-A to block repression of miR-26b-5p by E₂ must also be a critical mechanism of cross-talk of progesterone via PR-A in the rescue of invasiveness.

The above results implicate miR-92a-3p and miR-26b-5p as targets in the cross-talk of progestin/PR-A with E₂/ER that mediate rescue of invasiveness in luminal breast cancer cells. The regulation of these two microRNAs by E₂ was inhibited by the nuclear estrogen antagonist, tamoxifen (Fig. S3), consistent with genomic regulatory mechanisms.

miR-92a-3p and miR-26b-5p regulate genes associated with invasiveness and metastasis in luminal breast cancer cells

To further study the roles of miR-92a-3p and miR-26b-5p in relation to gene expression and metastasis, we generated two types of pooled (to avoid clonal bias) stable recombinant BT474 cells. In one case, miR-92a-3p (E₂-activated miRNA) was constitutively expressed (miR-92a-3p-On cells in Fig. 4A). In the other case, miR-26b-5p (E₂-repressed miRNA) was constitutively inhibited due to expression of a miR-26b-5p inhibitor (miR-26b-5p-Off cells in Fig. 4B). Hormonal regulation of miR-26b-5p and miR-92a-3p was unaffected in miR-92a-3p-On cells and miR-26b-5p-Off cells, respectively, indicating that these two miRNAs are regulated independently (Fig. 4, A and B). Both the miR-92a-3p-On cells (Fig. 4C) and the miR-26b-5p-Off cells (Fig. 4D) showed repressed invasiveness even in the absence of hormones, similar to the E₂-treated control cells (harboring Lenti-miR-blank plasmid). Moreover, the ability of R5020 to rescue invasiveness was lost in both the miR-92a-3p-On cells (Fig. 4C) and in the miR-26b-5p-Off cells (Fig. 4D), in contrast to the control cells. The miR-92a-3p-On cells and the miR-26b-5p-Off cells showed in vitro proliferation rates similar to that of the control cells (Fig. S4).

Next, the miR-92a-3p-On and miR-26b-5p-Off cells, treated with both E₂ and R5020, were examined for quantitative changes in the metastasis/invasion transcriptome, using a commercial pathway cDNA TaqMan PCR array. When compared with the control cells, both of the recombinant cells showed broad and partially overlapping increases in the expression of cellular adhesion molecules and inhibitors of metastasis but not a remarkable effect on the expression of genes known to promote metastasis (Fig. 4E; values in Table S6). The functionality of genes from the metastasis gene array was validated by individually knocking down three of the genes (RhoC, TIMP4, and MMP9), in BT474 cells as well as T47D cells. Both RhoC and MMP9 are known to be promoters of metastasis, and knocking down either one of these genes decreased invasive potential (Fig. 4, F and G). In contrast, TIMP4 is a known inhibitor of metastasis, and knocking down this gene increased invasiveness (Fig. 4, F and G). It may be noted, however, that our results show that the broader effects of regulating miR-92a-3p and miR-26b-5p on the transcriptome have the net effect of strongly regulating the invasive and metastatic phenotype. Therefore, miR-92a-3p and miR-26b-5p are part of independently regulated but convergent pathways through which E₂ controls genes that have functional roles in metastasis. It follows that the ability of PR-A to oppose regulation of these miRNAs by E₂ enables progesterone to induce a gene-regulatory pattern that supports metastasis.

An additional relevant question was whether estrogen and/or progesterone could at least partially regulate epithelial versus mesenchymal characteristics of luminal breast cancer cells, although it has been well-established that ER expression confers the epithelial phenotype. Using a commercial EMT panel antibody kit and BT474, T47D, and ZR-75-1 cell lines, we observed no change in established epithelial/mesenchymal markers when cells were treated individually or in combination with E₂ and R5020 (Fig. S5, A and B) in contrast to depletion of the ER apoprotein (Fig. S5C). Therefore, it was irrelevant to explore EMT in the context of hormonal regulation of miR-92a-3p or miR-26b-5p.

miR-92a-3p and miR-26b-5p profoundly impact metastasis in vivo

Studies of hormonal regulation of metastasis of model luminal breast tumor xenografts in mice are technically challenging for the following reasons. First, estrogen needs to always be present for the tumors to grow, optimally administered to the mice through implanted slow-release, low-dose E₂ pellets; therefore, the hormone levels cannot be modulated. Second, the constant exposure to even the low dose of E₂ needed to support tumor growth causes urinary retention and cystitis in the mice, limiting the duration of the experiments to about 2–3 weeks. Finally, luminal breast tumors are inherently less aggressive and metastasize relatively slowly, so that it is not possible to histologically detect micrometastases within the 2–3-week duration of the experiments. We overcame these problems in the current study design that investigated whether modulation of miRNAs 92a-3p and 26b-5p could govern metastasis in mice bearing luminal breast tumor xenografts.

We used as xenograft models the recombinant BT474 cells in which cross-talk between E₂/ER and progestin/PR-A affecting invasiveness was disrupted by constitutive expression of miR-92a-3p (miR-92a-3p-On cells discussed above) or constitutive inhibition of miR-26b-5p (miR-26b-5p-Off cells discussed above) for comparison with the control cells. As a metastasis assay validation tool, we also generated and used as xenografts pooled (to avoid clonal bias) stable recombinant BT474 cells in which PR was knocked down (BT474 PR-shRNA cells); in these cells, we confirmed that the PR knockdown resulted in loss of the ability of R5020 to rescue in vitro invasiveness from E₂ regulation (Fig. S6). The previously established range of plasma estrogen in mice implanted with the slow-release E₂ pellets is 1.8–4.8 nm (37, 38). The established range of plasma progesterone in mice on a commercial diet is 3.2–5.6 nm (38). To measure metastasis of subcutaneous implantation of the xenografts in the right flank, we measured tumor cell infiltration in the ipsilateral axillary lymph nodes by the sensitive assay of measuring mRNA for human GAPDH present in the lymph nodes, 14 days after implanting the xenografts. The control BT474 cells consistently infiltrated the lymph node, whereas depletion of PR in these cells showed virtually complete suppression of lymph node infiltration, validating the metastasis assay (Fig. 5, A and B). In mice bearing xenografts of miR-92a-3p-On cells (Fig. 5, C and D) or miR-26b-5p-Off cells (Fig. 5, E and F), metastasis was dramatically suppressed, demonstrating that the profound effects of changing the levels of these two miRNAs on cellular invasiveness observed above extend to their effects on metastasis in vivo.

The foregoing results strongly support the premise that positive (for miR-92a-3p) or negative (for miR-26b-5p) regulation of these two miRNAs by E₂ causes suppression of not only invasiveness but also metastasis of luminal breast cancer cells and that, by extension of this reasoning, the restoration of these miRNAs to their original levels (as in the control tumors) by progesterone via PR-A must support metastasis.

In primary human luminal breast tumors PR-A expression correlates negatively with miR-92a-3p and positively with miR-26b-5p

Using total RNA samples extracted from 53 ER+/PR+ primary luminal breast tumor specimens, the expression profile of PR-A mRNA was obtained and compared with the expression profiles of miR-92a-3p and miR-26b-5p obtained from the same RNA preparations. Despite the inherent and variable heterogeneity among the tumor specimens due to a variable ratio of tumor cells to stroma, miR-92a-3p correlated negatively with PR-A mRNA with an r = −0.272 (p = 0.004) (Fig. 6A), and miRNA-26b-5p correlated positively with PR-A mRNA with r = 0.342 (p = 0.001) (Fig. 6B). Moreover, as shown in the heat map in Fig. 6C, the inverse expression trends of the two miRNAs generally occurred within the same samples. Finally, relative PR-A protein levels among the tumors generally corresponded to the relative PR-A mRNA levels (Fig. 6D) with the caveat that the protein was measured only semiquantitatively by non-linear densitometry from Western blots (Fig. 2A) and was also necessarily extracted from a different part of each tumor specimen than RNA.

Cell line models and breast tumor specimens

BT474, T47D, and ZR-75-1 breast cancer cells were purchased from American Type Culture Collection. T47D-A (ER+/PR-A+/PR-B-null) and T47D-B (ER+/PR-B+/PR-A-null) recombinant cells isogenic with parental T47D cells were a generous gift from Dr. Katherine Horowitz (University of Colorado, Denver, CO) and were cultured as described previously (36). T47D-PR-A++ cells were previously generated in our laboratory (13). Recombinant BT474 cell lines with stable expression of PR shRNA (PR-shRNA cells), miR-92a-3p (miR-92a-3p-On cells), or miR-26b-5p inhibitor (miR-26b-5p-Off cells) and control cells harboring Lenti-miR-Blank plasmid were generated using PR shRNA lentiviral plasmid (catalog no. 0000436004, Sigma-Aldrich), hsa-miR-92a-3p miRNA Lentivector (catalog no. mh11076, ABM, Vancouver, Canada), or Lenti-miR-Off-hsa-miR-26b-5p vector (catalog no. mh30381, ABM) or control Lenti-miR-blank vector (catalog no. m007, ABM) by lentiviral transduction methods described below. All human tumor samples, classified as ER+/PR+ ductal carcinoma, were obtained from the Cooperative Human Tissue Network.

Cell culture and hormone depletion

Cell line models were cultured in DMEM supplemented with FBS (10%), penicillin (100 unit/ml), streptomycin (100 μg/ml), and l-glutamine (2 mm) at 37 °C in 5% CO₂. To maintain selection pressure, the medium for the recombinant BT474 cells contained 0.5 μg/ml puromycin, and the medium for T47D-A and T47D-B cells contained 200 μg/ml Geneticin. For hormone depletion, cells were plated at 30% confluence in phenol red-free medium supplemented with charcoal-stripped FBS and incubated at 37 °C in 5% CO₂ for 48 h.

Western blotting of cells and breast tumor tissues

Cell lysates were prepared as described (13). To prepare tumor tissue lysates, tissue (100 mg) suspended in 500 μl of radioimmune precipitation buffer was homogenized using the PRO200 tissue homogenizer (catalog no. 01-01200, BioGen, Cambridge, MA) for 15 s on ice and centrifuged at 15,000 × g, and supernatant was used. Western blotting was performed as described previously (13). The antibody probes include monoclonal rabbit anti-PR antibody (catalog no. 8757, Cell Signaling, Danvers, MA), EMT sampler kit antibodies (catalog no. 9782, Cell Signaling), or mouse monoclonal anti-GAPDH antibody (sc-4472, Santa Cruz Biotechnologies) and appropriate horseradish peroxidase–conjugated secondary antibodies (Vector Laboratories). Relative protein expression was determined by semiquantitative densitometry of autoradiographic film using ImageJ software (National Institutes of Health).

Boyden chamber transwell invasion assay

Cells (1 × 10⁵) were resuspended in the appropriate culture medium devoid of serum and phenol red and added to the top chamber of the FluoroBlok inserts (catalog no. 351152, 8-μm pore membrane; BD Biosciences) coated with growth factor reduced Matrigel (0.2 mg/ml). The chemoattractant comprised phenol red-free medium supplemented with FBS (20%). The appropriate hormone treatment was included in both the top and bottom chambers. Each treatment was replicated in three wells, and the entire experiment was repeated at least three times. Cells were allowed to invade for 24 h at 37 °C with 5% CO₂. Cells that invaded to the bottom surface were stained with calcein AM (4 μg/ml) in serum-free medium in the dark for 1 h at 37 °C with 5% CO₂. Images were captured in an identical manner from each well in five non-overlapping fields (the middle of the well and surrounding fields) using a ×4 objective. Images were analyzed using ImageJ software (National Institutes of Health), and the number of cells invaded was quantified by brightness and pixel size.

Isolation and measurement of microRNA and mRNA

Total RNA was isolated from cells or tissues using the miRCURY total RNA isolation kit (Exiqon, Vedbaek, Denmark). Breast tumor tissue lysates were prepared by suspending 50 mg of tissue in 500 μl of the lysis buffer and homogenized using the PRO200 tissue homogenizer (catalog no. 01-01200, BioGen) for 15 s on ice. Homogenized solution was centrifuged at high, 15,000 × g, and supernatant was used for RNA extraction. Reverse-transcription PCRs were performed using high-capacity complementary DNA archive kit or miRNA reverse transcription kit (Life Technologies, Inc., Carlsbad, CA). cDNA was measured by quantitative real-time RT-PCR using TaqMan probes and the StepOne Plus real-time PCR system (Life Technologies). All RNA measurements were performed in biological triplicates, and all Ct values were normalized to intrasample GAPDH (mRNA) or U6 snRNA (miRNA). RNA values were represented as -fold difference, which is calculated using the expression, 2^−ΔΔCt, where ΔΔCt = ΔCt sample − ΔCt calibrator.

Affymetrix profiling of microRNAs regulated by PR-A

Hormone-depleted T47D-A and T47D-B cells were treated with vehicle or R5020 (1 nm). Total RNA samples, isolated using the Exiqon miRCURY isolation kit, were analyzed at the University of Michigan Microarray Core using the Affymetrix miRNA microarray generation IV (Affymetrix, Santa Clara, CA). Expression values were normalized using quantile normalization with background subtraction. Log transformation to base 2, followed by one-way analysis of variance, was used to determine error. The differentially expressed genes were identified by comparing R5020 treatment with vehicle treatment (repressed or activated with a cutoff -fold difference of 1.5 and a p value < 0.05). Real-time RT-PCR was performed as described above to validate miRNAs that were exclusively regulated by PR-A.

Lentiviral transduction

Packaging of lentiviral particles with pCDH empty vector plasmid, pCDH-PR shRNA, hsa-miR-92a-3p miRNA Lentivector, or Lenti-miRa-Off-hsa-miR-26b-5p vector and transduction of cells were performed as described (13). The pool of recombinant cells was selected by culturing in puromycin-containing medium for 6 weeks.

Transfection of siRNA or miRNA inhibitor

Cells were plated to 30% confluence without antibiotic in phenol red–free DMEM supplemented with 10% charcoal-stripped FBS. 24 h later, cells were transfected with siRNA directed against specific gene targets, miRNA inhibitors (miRVana, Life Technologies) directed against specific miRNA targets, or appropriate non-silencing controls using Lipofectamine (Life Technologies).

Metastasis pathway gene array expression analysis

The human tumor metastasis fast TaqMan real-time PCR array (Life Technologies, Carlsbad, CA) was performed on StepOne Realtime (Applied Biosystems). cDNA samples from BT474 cells (control transduced cells), BT474 92a-3p-On cells, and BT474 miR-26b-5p-Off cells were analyzed. The ΔΔCt method was used as described above to quantify gene expression.

Mouse metastasis model

Female athymic nude mice (Envigo, Indianapolis, IN) were implanted with 0.72-mg slow-release estradiol pellets (Innovative Research of America, Sarasota, FL) on day 0. On day 3, 1 × 10⁷ to 2 × 10⁷ cells were suspended in 300 μl of equal parts DMEM and Matrigel and implanted subcutaneously in the right flank just below the right shoulder. Mice were sacrificed on day 17. Tumors, ipsilateral and contralateral axillary and inguinal lymph nodes, and livers were harvested from the mice. All tissues were homogenized, and total RNA was extracted as described above for human tissues. The degree of tissue infiltration by the human tumor cells (metastasis) was measured in terms of the amount of mRNA present for human GAPDH by quantitative real-time RT-PCR using human and mouse species-specific high efficiency Taqman probes. The mRNA values for human GAPDH were normalized to the mRNA for mouse GAPDH.