Hypoxia-inducible factor 1 is activated by dysregulated cyclin E during mammary epithelial morphogenesis

Abstract

Increased cyclin E expression has been identified in human tumors of diverse histologies, and in studies of primary breast cancers, high cyclin E is associated with poor prognosis. We have studied dysregulated cyclin E in epithelial tissues using organotypic cultures of human mammary epithelial cells and a murine model. We unexpectedly discovered that dysregulated cyclin E impairs normal acinar morphogenesis in vitro, and this is associated with the induction of p21(Cip1), p27(Kip1), and cellular senescence. Cyclin E-induced morphogenesis arrest is dependent upon hypoxia-inducible factor 1α (HIF-1α), which itself is induced by high cyclin E both in cultured mammary acini and in mammary epithelial tissues in a mouse model of deregulated cyclin E expression. We next determined that E2F activity directly regulates and is required for induction of HIF1A by cyclin E. Additionally, we found that cyclin E deregulation in mammary acini decreases, in an E2F-independent manner, expression of the EGLN1 prolyl hydroxylase that regulates HIF-1α degradation within the VHL ubiquitin ligase pathway. Together, our findings reveal a direct link between cyclin E and HIF-1 activities in mammary epithelial cells and implicate HIF-1 as a mediator of proliferation-independent phenotypes associated with high cyclin E expression in some human breast cancers.

Figures

Fig. 1.

Dysregulated cyclin E expression in MCF-10A cells impairs acinar morphogenesis in organotypic cultures. (A) MCF-10A cells transduced with the indicated retroviral constructs were seeded on basement membrane extract (Matrigel)-coated chamber slides. Cultures were grown for a total of 8 days, after which acinar structures were photographed (×11.25 magnification; insets display representative images), and acinar surface area was measured using ImageJ software. Graphs represent distribution of structures, with individual acinar measurements binned according to the indicated surface areas. Calculated P values for differences between control (vector) acinar size distribution and those of wild-type cyclin E (wt) and cyclin E(T380A) acini are 0.65 and 0.006, respectively. (B) MCF-10A cells transduced with control or cyclin E(T380A)-expressing retroviral vectors were grown in Matrigel for 18 days. (C) Representative acini were fixed after 18 days and stained for KI67 (green), GM130 (red, Golgi stain), and TOPRO (blue, nuclear counterstain). Arrowhead indicates arrested cyclin E(T380A) structure, and other cyclin E(T380A) structures shown are representative of the hyperproliferative acini. (D) (Left) Protein extracts were prepared from day 8 acinar cultures and immunoblotted for cyclin E. GRB2 expression is shown as loading control. (Right) Images are shown of representative control and arrested and hyperproliferative cyclin E(T380A) acini, fixed after 18 days of growth and stained for cyclin E (red) and TOPRO. (E) Tabulated BrdU incorporation results are shown for MCF-10A cells that were transduced with the indicated constructs and grown for 8 days in either acinar or monolayer cultures. (F) (Top) Representative (magnification, ×20) images are shown of ethidium bromide staining of live, day 8 acini, comprised of cells expressing the indicated constructs, for in situ cell death detection. (Bottom) Tabulated cleaved caspase 3 immunofluorescence (IF) detection results are displayed for fixed, day 18 acini. Smaller cyclin E(T380A) structures are those comprised of 12 or fewer cells per image, and larger structures are those with 13 or more. (G) Endogenous LC3 processing was measured in the indicated mammary acinar cultures, treated with either bafilomycin A1 for the indicated times or vehicle alone, and the accumulation of LC3-II was measured. Values displayed indicate average increase in normalized LC3-II signal after treatment, in two independent experiments. SD, standard deviation. (H) Representative transmission electron micrograph images of an intact, control MCF-10A acinar structure (magnification, ×400) or an arrested, cyclin E(T380A) structure (magnification, ×1,000), following growth in Matrigel. (I) (Top) HP1γ and/or SA β-Gal staining was performed for acinar cells, disaggregated from the indicated acinar cultures, or for cells grown in a monolayer. Representative results with staining for SA β-Gal (magnification, ×11.25) and nuclear HP1γ (magnification, ×100) are shown. Arrowheads indicate rare positive cells detected in monolayer cultures. (Bottom) The graph displays average fraction of cells positive for either SA β-Gal or HP1γ per field. Error bars indicate standard deviations.

Fig. 2.

Dysregulated cyclin E induces HIF-1α during acinar morphogenesis. (A) MCF-10A cells transduced with control or cyclin E(T380A)-expressing vectors were cultured for acinar morphogenesis over 8 days, formalin fixed and paraffin embedded (FFPE), and stained for detection of p21 and p27 (magnification, ×40, representative images [left]). (Right) Cells positive for either p21 or p27 are expressed as ratios to total cell number per ×40 magnification field. (B) Extracts were prepared from the indicated acinar cultures after 8 days of growth and immunoblotted for p21, p27, and GRB2 proteins. Relative abundances of p21 and p27 (normalized to GRB2 signal) are shown as averages from three independent experiments (tripl.) along with calculated standard deviations. (C and E) RNA was extracted from monolayer or acinar cultures after 8 days of culture growth and analyzed by quantitative RT-PCR for the expression of CDKN1A (p21) and CDKN1B (p27) (C) or the indicated HIF-1-regulated genes (E). Quantitative RT-PCRs were performed in triplicate, and the data shown represent the average from at least two different experiments. Error bars show standard deviation. (D) Shown are GSEA output data generated from comparison of gene expression signatures of CD71high Ter119high bone marrow cells from cyclin ET74A T393A mice, compared to those from wild-type mice. A signature corresponding to dysregulated expression of predicted HIF-1 targets is shown along with the corresponding P value and false-detection rate q value (FDR q) (48). (F) (Top) MCF-10A acini were FFPE processed as described above for panel A and stained for HIF-1α detection. Smaller structures are those comprised of 12 or fewer cells per section, and larger structures are those with 13 or more. Acini considered positive for HIF-1α staining contain at least one positive cell. Inset, representative image of a HIF-1α-positive structure is shown (magnification, ×40). (Bottom) Extracts prepared from the indicated acinar cultures after 8 days of growth were immunoblotted for HIF-1α and GRB2 proteins, and relative abundance of HIF-1α was quantified in four independent experiments. (G and H) HIF1A or HIF2A gene expression for the indicated acini or monolayer cultures was measured as described above.

Fig. 3.

HIF-1 induction occurs in primary mammary epithelial cells of cyclin ET74A T393A knock-in mice. (A) Expression of HIF-1 target genes was measured as described in the legend to Fig. 2C in freshly isolated mammary epithelial cells from four cyclin ET74A T393A knock-in mice versus wild-type mammary epithelial cells (obtained from age-matched, day 15 p.c., no. 4 mammary glands). (B) HIF-1α, p21, p27, and GRB2 (loading control) protein levels were assayed by immunoblotting protein extracts prepared from wild-type and knock-in mammary epithelial cells. (C) Transcript levels of HIF1A, CDKN1A, and CDKN1B were measured as described above for panel A.

Fig. 4.

Impaired morphogenesis of cyclin E(T380A) acini is dependent on HIF-1α. (A) MCF-10A cells first transduced with control or cyclin E(T380A)-expressing constructs were again transduced with vectors expressing a short hairpin targeting HIF1A (sh-HIF1A) or luciferase (sh-ctrl). (Left) Protein lysates were prepared from monolayer MCF-10A cells transduced with the indicated vectors and then subjected to low oxygen for 16 h. HIF-1α immunoblot to determine gene knockdown efficiency is shown. (Right) Acini from cells transduced with the indicated constructs were grown for 8 days, and then protein extracts were prepared and immunoblotted for cyclin E, HIF-1α, p21, p27, and GRB2. (B) Gene expression of HIF1A, PGK1, CDKN1A, and CDKN1B was measured from the indicated cultures by quantitative RT-PCR. (C) Individual cells were isolated from the indicated acini and stained for HP1γ as described in the legend to Fig. 1E. (D) Gene expression of VHL and PGK1 was measured from the indicated cultures by quantitative RT-PCR. (E and F) MCF-10A cells transduced with the indicated constructs were grown in mammary acinar cultures, and epithelial structure sizes were measured after 8 days of growth as described in the legend to Fig. 1A. Calculated P values for differences in size distributions between both control and sh-VHL structures and control and sh-HIF1A were <0.001. (G) Knockdown efficiency of CDKN1A and CDKN1B was assayed with the indicated cultures by quantitative RT-PCR. (H) MCF-10A cells transduced with the indicated constructs were grown in mammary acinar cultures, and epithelial structure sizes were measured after 8 days of growth as described in the legend to Fig. 1A. Calculated P value for difference in size distribution of control and p21/p27 knockdown acini, <0.001.

Fig. 5.

HIF1A is a direct transcriptional target of E2F. (A) Expression of the indicated E2F target genes was analyzed using RNA prepared from day 8 acini. Insets show abundance of total and phosphorylated Rb protein in extracts made from the indicated acinar cultures. (B) (Top) Schematic representation of the 5′ end of the HIF1A gene. The region spanning nucleotides −2180 to +28 (relative to the transcription start site) was cloned for use in all reporter assays. The gray box represents nucleotides −255 to −56, which contain six predicted E2F binding motifs indicated by the black bars. The dark box (*) represents positions −89 to −74, containing overlapping, predicted E2F binding motifs that were mutated in experiments described in the legend to panel D. (Bottom) Nucleotide sequence of the E2F-binding site-containing region within HIF1A, with positions −89 to −74 indicated in bold typeface. (C) (Top) 293T cells transiently transfected with expression plasmids for the HIF1A promoter reporter, Renilla luciferase, and either E2F-1 or the E2F-1-E132 mutant were assayed for the activation of the HIF1A reporter. (Bottom) MCF-10A cells stably expressing HIF1A promoter reporter and the indicated E2F-expressing constructs were plated for acinar morphogenesis. Following 8 days, luciferase activity was measured. Error bars indicate standard deviations from three separate experiments. Insets show expression of wild-type E2F-1 and the E132 mutant. (D) 293T cells were transfected with expression plasmids for wild-type E2F-1, Renilla luciferase, and the wild-type HIF1A promoter reporter, a deletion mutant that removes the −255 to −56 region (Δ), or another mutant reporter containing nucleotide substitutions within positions −89 to −74 (*). Reporter activities are displayed relative to the activation of the wild-type reporter by E2F-1. (E) MCF-10A cells stably expressing the indicated HIF1A promoter reporters and cyclin E(T380A)-expressing or empty (−) vector were plated for acinar morphogenesis. Following 8 days, luciferase activity was measured, and change in reporter activation with cyclin E(T380A) expression was normalized to the control's activity level within each reporter set. (F) Quantitative ChIP analysis of endogenous E2F-1 occupancy at the HIF1A promoter versus a 10-kb-upstream region was performed with MCF-10A cells transduced with the indicated constructs. Normal mouse IgG was used for the control immunoprecipitations. Average values from two different experiments are shown. (G) Cyclin E kinase and endogenous CDK2 activities using purified histone H1 as substrate were measured for protein extracts prepared from the indicated MCF-10A acini. Activity levels were quantified from autoradiograms using ImageJ and are expressed relative to those measured in control acini. (H) MCF-10A cells stably expressing the HIF1A promoter reporter were transduced with the indicated vectors, then grown in morphogenesis cultures for 8 days, and assayed for transactivation of the reporter as indicated. Insets show expression of cyclin E and the E2F-1-E132 mutant.

Fig. 6.

Dysregulated cyclin E downregulates EGLN1 expression in mammary acini. (A) Epithelial cells were isolated from day 15 p.c. wild-type (wt) and cyclin ET74A T393A mammary glands. Protein extracts were prepared and immunoblotted for cyclin E, EGLN1, and GRB2 (left), with quantitation shown for three independent experiments, and RNA was purified for EGLN1 transcript analysis. (B) (Left) Protein extracts were prepared from day 8 acini, and cyclin E, EGLN1, and GRB2 expression was measured by immunoblotting. (Right) Analysis of EGLN1 expression is shown from monolayer cells or day 8 MCF-10A acini transduced with the indicated constructs. (C) RNA was prepared from day 8 MCF-10A acini comprised of cells transduced with the indicated vectors, and the expression of HIF1A and EGLN1 was measured as described above. (D) MCF-10A cells were transduced with the indicated retroviral constructs. Protein extracts from day 8 acini were immunoblotted for EGLN1, HIF-1α, and GRB2. (E) RNA prepared from MCF-10A acini transduced similarly to those described in the legend to panel D was analyzed for the expression of CDKN1B, VEGFA, and SCL2A1.

Fig. 7.

Schematic of the proposed cooperative mechanisms by which dysregulated cyclin E regulates HIF-1α expression in mammary epithelial cells during morphogenesis.