NEMO, a Transcriptional Target of Estrogen and Progesterone, Is Linked to Tumor Suppressor PML in Breast Cancer

Abstract

The beneficial versus detrimental roles of estrogen plus progesterone (E+P) in breast cancer remains controversial. Here we report a beneficial mechanism of E+P treatment in breast cancer cells driven by transcriptional upregulation of the NFκB modulator NEMO, which in turn promotes expression of the tumor suppressor protein promyelocytic leukemia (PML). E+P treatment of patient-derived epithelial cells derived from ductal carcinoma in situ (DCIS) increased secretion of the proinflammatory cytokine IL6. Mechanistic investigations indicated that IL6 upregulation occurred as a result of transcriptional upregulation of NEMO, the gene that harbored estrogen receptor (ER) binding sites within its promoter. Accordingly, E+P treatment of breast cancer cells increased ER binding to the NEMO promoter, thereby increasing NEMO expression, NFκB activation, and IL6 secretion. In two mouse xenograft models of DCIS, we found that RNAi-mediated silencing of NEMO increased tumor invasion and progression. This seemingly paradoxical result was linked to NEMO-mediated regulation of NFκB and IL6 secretion, increased phosphorylation of STAT3 on Ser727, and increased expression of PML, a STAT3 transcriptional target. In identifying NEMO as a pivotal transcriptional target of E+P signaling in breast cancer cells, our work offers a mechanistic explanation for the paradoxical antitumorigenic roles of E+P in breast cancer by showing how it upregulates the tumor suppressor protein PML. Cancer Res; 77(14); 3802-13. ©2017 AACR.

Figure 1

Only a subset of patient-derived ER+/PR+ DCIS cells responded to hormone treatment in vitro by increasing mammosphere formation efficiency. (A) Only a subset of cases responded to steroid hormone treatment in vitro by increasing mammosphere-forming efficiency (cases 7–11). Data are presented as mean± SEM. (*indicates E+P increased mammosphere efficiency compared to vehicle control, P<0.05). (B) Representative IF images of primary mammospheres from three patient biopsies embedded in paraffin. FSP1 is shown in green (Fibroblast marker), K5/K19 in red (Epithelial marker), and Hoechst in blue, demonstrating the appearance of stromal (FSP1 positive), epithelial (keratin positive), and mixed stromal and epithelial mammospheres (mixed). (C) Bar graph representing quantification of epithelial-derived, stromal derived, and mixed epithelial and stromal derived mammospheres, comparing E+P treated to control. The data represent the mean ± SEM (n=3, *P<0.05).

Figure 2

E+P treatment of patient derived DCIS cells results in increased mammosphere efficiency in vitro and increased IL-6 expression as a function of invasive progression in patients’ DCIS. (A) Graph depicts mammosphere formation efficiency in representative cases of non-responders (3 total cases examined) and a responder (1 total case examined), and (B) corresponding IL-6 secretion as measured by ELISA of the cell culture media (*P<0.05 compared to vehicle control). (C) Boxplots showing the distribution of IL-6 expression from 30 cases of pure DCIS and 60 cases of DCIS with IDC. A two-sample t-test was used to compare expression in pure DCIS to expression in DCIS with associated with IDC. Expression was higher in DCIS with associated IDC compared to pure DCIS. On DCIS with associated IDC samples, IL-6 expression was also significantly higher when comparing DCIS regions to the corresponding IDC regions using a paired t-test. IL-6 expression levels were quantified using Metamorph. (D) Representative images of pure DCIS stained for IL-6 (green) and K-5 and-19 (red). (E) Representative images from patients with both DCIS (left) and IDC (right), stained for IL-6 (green) and K-5 and-19 (red).

Figure 3

(A) Cells from patient samples of atypical hyperplasia and DCIS were cultured under non-adherent conditions for 1 week in the presence of vehicle, E (10nM), P (100nM), or E+P. NEMO and IL-6 gene expression was measured by qPCR. (B) ER+PR+ breast cancer cells were cultured as monolayers for 12 hours in the presence of vehicle, E (10 nM), P (10nM), or E+P. NEMO gene expression was measured by qPCR. Data are presented as mean± SEM. * P < 0.05 compared to vehicle control. ChIP assays were performed on MCF7 breast cancer cells (C) and on primary patient DCIS with IDC (D) treated with vehicle, E (10nM), P (10nM), or E+P for 1h. Immunoprecipitation was performed using an anti-ER antibody and qPCR was performed using primers targeting the potential ERE within the IKBKG promoter region. Data is presented as mean± SEM. * P<0.05 compared to IgG control.

Figure 4

NEMO is required for the E+P-induced increase in NFκB activation and IL-6 secretion in ER+PR+ breast cancer cells. (A) Western analysis was used to confirm knock down of NEMO in MCF7 and BT474. NT: non-transduced control. NS: scrambled non-silencing control. KD: NEMO-targeting shRNA. (B) MCF7 and BT474 cells were serum-starved for 24h before treatment with 10nM E and 10nM P. Nuclear extract was collected 48 h later and NFκB p65 activity within the nucleus was measured. * P<0.05 compared to vehicle (V) control. (C) MCF7 and BT474 cells were serum-starved for 24 h before treatment with 10nM E and 10nM P. Cell culture media was collected 48h later and IL-6 secretion was measured by ELISA. * P<0.05 compared to vehicle (V) control.

Figure 5

NEMO-KD DCIS lesions showed increased in vivo invasive progression. (A) MIND is a transplantation technique that involves intraductal injection of DCIS cells into the primary mouse mammary ducts (a). (b) Normal mammary ducts include a single layer of ductal epithelium surrounded by a layer of myoepithelium. (c) Ductal carcinoma in situ (DCIS) refers to in situ or non-invasive breast cancer in which cancer cells are contained within the boundaries of the myoepithelial layer and the basement membrane. (d) Invasive DCIS refers to DCIS lesions in which the cancer cells have bypassed the normal boundaries of the myoepithelial layer and have invaded the surrounding stroma. Invasive DCIS is assessed by counting the number of invasive DCIS lesions per high power field as well as the maximum distance traveled by the cancer cells beyond the mammary ductal system; indicated by the red bracket in (d). Glands from control and NEMO-KD MCF7 (B and C) and ER+/PR+ DCIS.com (D and E) MIND xenografts were collected at 6 and 8 weeks post-intraductal injection, respectively. IF staining of K5/19 (red), SMA (green), and hoechst (blue) in MCF7 (B) and ER+/PR+ DCIS.com (D) MIND demonstrating how distance of invasion was measured. (C and E) Bar graphs represent the maximum distance of invasion and number of invasive lesions in control and NEMO KD for MCF7 (C) and ER+/PR+ DCIS.com (E) MIND xenografts. Data represent the mean ± SEM. (n=4, *P<0.05).

Figure 6

NEMO-KD DCIS lesions showed a significant reduction in IL-6 signaling and PML expression. Glands from control and NEMO-KD MCF7 MIND xenografts were collected at 6 weeks post-intraductal injection and expression of different markers in NEMO-KD MIND xenografts were measured. (A) IF images of control (a,c,e,g, and i) and NEMO-KD MCF7 (b,d,f,h, and j) xenografts stained with IL-6 (a and b) shown in green, pSTAT3 (c and d) in red, PML (e and f) in red, RIPK1 (g and h) in red and pERK (i and j) in red, K19 shown in green, and hoechst in blue. Scale bars=50μm (10μm for PML images), 40x magnification. The white arrows point to the PML nuclear bodies (B) Bar graphs of fluorescence intensity units for IL-6 (a), pSTAT3 (b), PML (c), RIPK1 (d) and p-ERK (e) in control and NEMO-KD MCF7 cells. Measurements were obtained by Metamorph. The data represent the mean ±SEM (n=4, *P<0.05).

Figure 7

An illustration of mechanism by which E+P may protect against aggressive breast cancer. E+P increases ER binding to the IKBKG promoter and results in increased NEMO expression. NEMO is the regulatory subunit of the IKK (inhibitor of NF-kappa-B kinase) core complex, which phosphorylates the inhibitor of NF-kappa-B (IκB) thus promoting the dissociation of the IκB and ultimately degradation of the inhibitor and subsequent activation and nuclear translocation of NFκB. NF-κB signaling promotes NFκB target gene transcription, including IL-6, and activation of IL-6/STAT3 signaling subsequently leading to the expression of a tumor suppressive protein, PML.