Schematic representation of <i>Mcl-1</i> gene showing approximate locations of ERE half-sites and Sp1 binding sites.

<p>A sequence analysis of the Mcl-1 promoter region showed that the Mcl-1 promoter region includes 5 ERE half-sites (black boxes). These half-sites are located at 3683 bp, 3376 bp, 2713 bp, 2554 bp and 1068 bp upstream of the translation start site. In addition, there are multiple Sp-1 transcription factor sites within the promoter (gray boxes). Three of the ERE half-sites, located at 3683 bp, 2713 bp and 2554 bp upstream of the translation start site, are in close proximity to Sp1 binding sites. These regions (regions 1–3) are denoted by black lines.</p

Estrogen fails to increase Mcl-1 protein expression in ERα- breast cancer cell lines.

<p>(A) Western blot analysis of SK-BR-3 cells was preformed following 24-hour stimulation with estrogen (10 nM). (B) Western blot analysis of MDA-MB-231 cells was preformed following 24-hour stimulation with estrogen (10 nM). In both experiments, cells were serum-starved for 5 days prior to treatment with estrogen. Blots were reprobed with anti-β-actin as a loading control. (C) Relative accumulation of Mcl-1 protein expression in SK-BR-3 cells, confirmed by densitometry. Data represents mean of two independent experiments ± standard error. (D) Relative accumulation of Mcl-1 protein expression in MDA-MB-231 cells, confirmed by densitometry. Data represents mean of two independent experiments ± standard error.</p

ERα binds to specific region within Mcl-1 promoter in MCF-7 cells.

<p>(A) Chromatin immunoprecipitation (ChIP) was performed using antibody specific to ERα. ChIP was performed 6 hours after estrogen (10 nM) treatment. Positive control (immunoprecipitation of RNA Polymerase II), negative control (immunoprecipitation of Normal Mouse IgG) and no antibody control (beads alone) are shown. Results represent fold enrichment values obtained by comparing cT values of ChIP samples to cT values of input. Data represents the mean of 3 independent experiments ± standard error. * indicates p≤0.001 compared to input; # indicates p≤0.2 compared to input. (B) PCR products were run on agarose gel to evaluate ChIP specificity. Primers specific to ERE half-site located 3683 bp upstream of translation start site of Mcl-1 were used.</p

Estrogen increases ERα binding to specific region on Mcl-1 promoter.

<p>(A) Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 1). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Pull-down products were analyzed using SDS/polyacrylamide gel electrophoresis and western blotting. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (B) Blot was probed with antibody specific for ERβ. (C) Blot was probed with antibody specific for Sp1. (D) Blot was probed with antibody specific for Sp3. (E) Relative accumulation of ERα protein expression, confirmed by densitometry. (F) Relative accumulation of Sp1 protein expression, confirmed by densitometry. (G) Schematic representation of <i>Mcl-1</i> gene showing approximate locations of biotin labeled probes used for Streptavidin pull-down.</p

Estrogen receptor fail to bind to other ERE half sites in the Mcl-1 promoter.

<p>Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 2). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Pull-down products were analyzed using SDS/polyacrylamide gel electrophoresis and western blotting. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (B) Blot was probed with antibody specific for Sp1. (C) Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 3). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (D) Blot was probed with antibody specific for Sp1.</p

Estrogen increases Mcl-1 protein expression in ERα+ breast cancer cell lines.

<p>(A) Western blot analysis of MCF-7 was performed following 24-hour stimulation with increasing concentrations of estrogen (10⁻² nM–10 nM). (B) Western blot analysis of ZR-75 was performed following 24-hour stimulation with increasing concentrations of estrogen (10⁻² nM–10 nM). In both experiments, cells were serum-starved for 5 days prior to treatment with estrogen. Blots were reprobed with anti-β-actin as a loading control. (C) Relative accumulation of Mcl-1 protein expression in MCF-7 cells was confirmed by densitometry. Data represents mean of three independent experiments ± standard error. (D) Relative accumulation of Mcl-1 protein expression in ZR-75 cells was confirmed by densitometry. Data represents mean of three independent experiments ± standard error.</p

Estrogen increases Mcl-1 mRNA expression.

<p>Real-time PCR analysis of Mcl-1 transcript levels in (A) MCF-7 and (B) ZR-75 was performed following 24-hour stimulation with estrogen (10 nM). In addition, Real-time PCR analysis of Mcl-1 transcript levels in (C) MCF-7 and (D) ZR-75 was performed following a short 6-hour time course with estrogen (10 nM). E) Finally, MCF-7 cells were treated with a range of estrogen concentrations (0.1 nM–10 nM) and Mcl-1 mRNA levels analyzed after 6 hours. In all experiments, 100 ng template RNA was amplified using primers specific to Mcl-1. qPCR results were standardized using primers for housekeeping gene cyclophilin or TATA box binding protein (TBP). Fold change represents the results relative to changes in basal levels observed in untreated sample. Data represents the mean of three independent experiments ± standard error. (* indicates p≤0.0002 compared to untreated control cells).</p

Knockdown of ERα results in decrease in Mcl-1 mRNA expression.

<p>(A) Western blot analysis confirms ERα silencing in MCF-7 cells after siRNA transfection. Blot was reprobed with anti-β-actin as a loading control. (B) Real-time PCR analysis of Mcl-1 transcript levels in MCF-7 cells was performed. Cells were serum starved for 5 days prior to transfection. After transfection, cells were stimulated with estrogen (E2, 10 nM) for 24-hours. For qPCR, 100 ng template RNA was amplified using primers specific to Mcl-1. qPCR results were standardized using primers for housekeeping gene cyclophilin. Results are expressed as fold change relative to changes in basal levels observed in untreated sample. Data represents the mean of three independent experiments ± standard error. (* indicates p≤0.001 compared to untreated control cells).</p

Treatment with anti-estrogen Tamoxifen and Fulvestrant decrease Mcl-1 protein expression.

<p>(A) Western blot analysis of MCF-7 cells was performed following 24-hour treatment with either Tamoxifen (200 nM) or Fulvestrant (500 nM) in combination with estrogen (10 nM). (B) Western blot analysis of ZR-75 cells was performed following 24-hour hour treatment with either Tamoxifen (200 nM) or Fulvestrant (500 nM) in combination with estrogen (10 nM). Blots were reprobed with anti-β-actin as a loading control. (C) Relative accumulation of Mcl-1 protein expression in (C) MCF-7 cells, and (D) ZR-75 cells confirmed by densitometry. This represents the trend in three independent experiments.</p

Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia

<p>Autophagy is an intracellular lysosomal degradation pathway where its primary function is to allow cells to survive under stressful conditions. Autophagy is, however, a double-edge sword that can either promote cell survival or cell death. In cancer, hypoxic regions contribute to poor prognosis due to the ability of cancer cells to adapt to hypoxia in part through autophagy. In contrast, autophagy could contribute to hypoxia induced cell death in cancer cells. In this study, we showed that autophagy increased during hypoxia. At 4 h of hypoxia, autophagy promoted cell survival whereas, after 48 h of hypoxia, autophagy increased cell death. Furthermore, we found that the tyrosine phosphorylation of EGFR (epidermal growth factor receptor) decreased after 16 h in hypoxia. Furthermore, EGFR binding to BECN1 in hypoxia was significantly higher at 4 h compared to 72 h. Knocking down or inhibiting EGFR resulted in an increase in autophagy contributing to increased cell death under hypoxia. In contrast, when EGFR was reactivated by the addition of EGF, the level of autophagy was reduced which led to decreased cell death. Hypoxia led to autophagic degradation of the lipid raft protein CAV1 (caveolin 1) that is known to bind and activate EGFR in a ligand-independent manner during hypoxia. By knocking down CAV1, the amount of EGFR phosphorylation was decreased in hypoxia and amount of autophagy and cell death increased. This indicates that the activation of EGFR plays a critical role in the switch between cell survival and cell death induced by autophagy in hypoxia.</p