PHLDA1 expression is controlled by an estrogen receptor-NFκB-miR-181 regulatory loop and is essential for formation of ER+ mammospheres

Abstract

Crosstalk between estrogen receptor (ER) and the inflammatory nuclear factor κB (NFκB) pathway in ER+ breast cancers may contribute to a more aggressive phenotype. Pleckstrin Homology-Like Domain, Family A, member 1 (PHLDA1), a target gene of ER-NFκB crosstalk, has been implicated in cell survival and stem cell properties. 17β-estradiol (E2), acting through ERα, and pro-inflammatory cytokines, acting through NFκB, increase the nascent transcript and PHLDA1 messenger RNA stability, indicating both transcriptional and post-transcriptional control of PHLDA1 expression. We show that PHLDA1 is a direct target of miR-181 and that mature miR-181a and b, as well as their host gene, are synergistically downregulated by E2 and tumor necrosis factor α, also in an ER- and NFκB-dependent manner. Thus, ER and NFκB work together to upregulate PHLDA1 directly through enhanced transcription and indirectly through repression of miR-181a and b. Previous studies have suggested that PHLDA1 may be a stem cell marker in the human intestine that contributes to tumorigenesis. Our findings that PHLDA1 is upregulated in mammospheres (MS) of ER+ breast cancer cells and that PHLDA1 knockdown impairs both MS formation and the expansion of aldehyde dehydrogenase (ALDH)-positive population, suggest that PHLDA1 may play a similar role in breast cancer cells. Upregulation of PHLDA1 in MS is largely dependent on the NFκB pathway, with downregulated miR-181 expression a contributing factor. Over-expression of miR-181 phenocopied PHLDA1 knockdown and significantly impaired MS formation, which was reversed, in part, by protection of the PHLDA1 3' untranslated region (UTR) or overexpression of PHLDA1 lacking the 3'UTR. Furthermore, we find that elevated PHLDA1 expression is associated with a higher risk of distant metastasis in ER+ breast cancer patients. Altogether, these data suggest that high PHLDA1 expression is controlled through an ER-NFκB-miR-181 regulatory axis and may contribute to a poor clinical outcome in patients with ER+ breast tumors by enhancing stem-like properties in these tumors.

Figure 1

PHLDA1 is regulated by E2 and TNFα at both transcriptional and post-transcriptional level. (a) PHLDA1 mRNA expression was measured by QPCR in RNA from MCF-7 cells treated for 2 hrs with vehicle control (Veh) or the combination of E2 (10 nM) and TNFα (10 ng/mL). siERα (50 nM) was transfected 48 hrs prior to E2 and TNFα treatment or IKK7 (1 μM) was added 1 hr prior to treatment. ***P<0.001 for siERα and IKK7 groups vs. control E2+TNFα group. (b) PHLDA1 protein was measured by Western Blot in MCF-7 cells treated for 16 hrs with Veh, E2, TNFα or the combination of E2 and TNFα. The inhibitors ICI or IKK7 (1 μM each) were added 1 hr prior to treatment. β-actin served as a loading control. Densitometry was performed and the numbers above each PHlDA1 band indicate protein expression relative to both β-actin and control group. (c) The level of nascent PHLDA1 transcripts was measured in MCF-7 cells treated for 2 hrs with Veh or E2 and TNFα. ICI or IKK7 was added 1 hr prior to treatment. ***P<0.001 for ICI and IKK7 vs. none in the presence of E2+TNFα. (d) PHLDA1 mRNA stability was measured in MCF-7 cells in the presence of Act D (1 μg/mL) added alone or in combination with E2 and TNFα treatment for up to 2 hrs.

Figure 2

PHLDA1 is a direct target of miR-181a and b family members. (a) PHLDA1 mRNA expression was measured in MCF-7 cells transfected with siNeg or mimics for miR-181a, miR-181b, or both (20 nM each) followed by E2 and TNFα treatment for 2 hrs. ***P<0.001 for each of the miR-181 mimic groups compared to siNeg control. (b) PHLDA1 protein was measured in MCF-7 cells transfected as in (a), followed by E2 and TNFα treatment for 16 hrs. (c) PHLDA1 protein was measured in cells transfected with different concentrations of PHLDA1-miR-181 target protector (0-1 μM) together with miR-181a and b mimics, followed by E2 and TNFα treatment for 16 hrs.

Figure 3

E2 and TNFα down-regulate miR-181A1 HG and mature miR-181a and b in an ER and NFκB dependent manner. (a) miR-181 HG mRNA was measured in MCF-7 cells treated with E2, TNFα, or the combination for 2 hrs. All treatment groups were significantly different than vehicle (Veh) control P<0.001. Treatment with E2+TNFα was significantly different from E2 or TNFα alone, *P<0.05, ***P<0.001. (b) ICI or (c) IKK7 was added 1 hr prior to E2 and TNFα treatment. *P<0.05, ***P<0.001, ns; not significant. miR-181 HG (d) and mature miR-181a (e) and mature miR-181b (f) were measured following treatment with E2 or E2+TNFα for up to 24 hrs. Significant differences between E2 and E2+TNFα are indicated. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

B>. Post-transcriptional regulation of PHLDA1 by endogenous miR-181a and b. (a) PHLDA1 mRNA was measured in cells transfected with or without the target protector (1 μM, 48 hrs) prior to Act D treatment for 2 hrs. **P<0.01. (b) PHLDA1 mRNA expression was measured in MCF-7 cells transfected with siNeg or anti-miR-181a and b inhibitors (A-miR, 100 nM each) followed TNFα treatment for 2 hrs. **P<0.01.

Figure 5

PHLDA1 is up-regulated while miR-181A1 HG and mature miR-181b are down-regulated in ER+ breast cancer cell MS. (a) PHLDA1 mRNA expression in standard adherent 2D culture of ER+ breast cancer cells was compared to MS culture. (b) PHLDA1 protein was measured in MCF-7 2D and MS cultures. (c) PHLDA1 mRNA expression in MS was analyzed after 6 hrs IKK7 treatment or 24 hrs ER antagonist treatment (1 μM each). (d) Expression levels of miR-181A1 HG and mature miR-181a and b were compared in 2D vs. MS culture. *P<0.05 or ***P<0.001 compared to 2D or vehicle (Veh) treated controls.

Figure 6

PHLDA1 is essential for MCF-7 MS formation and expansion of ALDH-positive cells. (a, b) MCF-7 cells were transfected with siNeg or siPHLDA1 (50 nM). After 48 hrs, cells were seeded at single-cell density in low attachment plates and MS were allowed to develop. After 7 days, the total number of MS ≥ 75 μm (a) and the average diameter of MS (b) were measured. ***P<0.001 compared to siNeg control. (c, d) MCF-7 cells transfected with siNeg or siPHLDA1 (50 nM each, 48 hrs) were treated with E2+TNFα for 2 hrs, washed and allowed to recover for 72 hrs prior to running the Aldefluor assay and FACS analysis. ***P<0.001 compared to siNeg control. In (c) quantitation of the ALDH-positive population is indicated. In (d) representative scatter plots from FACS are shown. (e) MCF-7 cells were transfected with miR-181a and b mimics (20 nM each), either alone or in combination with the PHLDA1-miR-181 target protector (1 μM). 48 hrs later, cells were seeded for MS and after 7 days the number of MS was measured. **P<0.01, ***P<0.001. (f) Stable cell lines overexpressing PHLDA1 or empty vector control were transfected with miR-181a and b mimics (20 nM each) and MS formation was measured. *P<0.05, ns, not significant.

Figure 7

PHLDA1 is associated with increased risk of distant metastasis in ER+ breast cancers. (a, b, c) Kaplan-Meier analysis demonstrating the association between PHLDA1 expression and distant metastasis free survival (DMFS) are indicated for (a) all breast cancer (BC) patients, (b) only for ER+ BC, and (c) only for ER− BC patients.

Figure 8

B>. Model for PHLDA1 regulation and function in ER+ breast cancer cells. ER and NFκB work together to regulate PHLDA1 through a direct transcriptional mechanism and an indirect, post-transcriptional mechanism that involves down-regulation of miR-181. High PHLDA1 expression and low miR-181a/b is essential for CSC-like properties. In turn, this may lead to an aggressive breast cancer phenotype and a poor patient outcome.