Jeong-Yeon Lee

Landscape of somatic mutations in 560 breast cancer whole-genome sequences

We analysed whole genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. 93 protein-coding cancer genes carried likely driver mutations. Some non-coding regions exhibited high mutation frequencies but most have distinctive structural features probably causing elevated mutation rates and do not harbour driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed 12 base substitution and six rearrangement signatures. Three rearrangement signatures, characterised by tandem duplications or deletions, appear associated with defective homologous recombination based DNA repair: one with deficient BRCA1 function; another with deficient BRCA1 or BRCA2 function; the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operative, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.

Anticancer effects of wogonin in both estrogen receptor-positive and -negative human breast cancer cell lines in vitro and in nude mice xenografts.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of wogonin in human breast cancer. Effects of wogonin were examined in estrogen receptor (ER)‐positive and ‐negative human breast cancer cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral wogonin was examined on tumor xenograft growth in athymic nude mice. The molecular changes associated with the biological effects of wogonin were analyzed by immunoblotting. Cell growth was attenuated by wogonin (50–200 μM), independently of its ER status, in a time‐ and concentration‐dependent manner. Apoptosis was enhanced and accompanied by upregulation of PARP and Caspase 3 cleavages as well as proapoptotic Bax protein. Akt activity was suppressed and reduced phosphorylation of its substrates, GSK‐3β and p27, was observed. Suppression of Cyclin D1 expression suggested the downregulation of the Akt‐mediated canonical Wnt signaling pathway. ER expression was downregulated in ER‐positive cells, while c‐ErbB2 expression and its activity were suppressed in ER‐negative SK‐BR‐3 cells. Wogonin feeding to mice showed inhibition of tumor growth of T47D and MDA‐MB‐231 xenografts by up to 88% without any toxicity after 4 weeks of treatment. As wogonin was effective both in vitro and in vivo, our novel findings open the possibility of wogonin as an effective therapeutic and/or chemopreventive agent against both ER‐positive and ‐negative breast cancers, particularly against the more aggressive and hormonal therapy‐resistant ER‐negative types.

DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression

DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown. Here we identify that DOT1L cooperates with c-Myc and p300 acetyltransferase to epigenetically activate epithelial–mesenchymal transition (EMT) regulators in breast cancer progression. DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions. The upregulation of these EMT regulators by the DOT1L-c-Myc-p300 complex enhances EMT-induced breast cancer stem cell (CSC)-like properties. Furthermore, in vivo orthotopic xenograft models show that DOT1L is required for malignant transformation of breast epithelial cells and breast tumour initiation and metastasis. Clinically, DOT1L expression is associated with poorer survival and aggressiveness of breast cancers. Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

Inhibitor of DNA Binding 1 Activates Vascular Endothelial Growth Factor through Enhancing the Stability and Activity of Hypoxia-Inducible Factor-1α

Inhibitor of DNA binding 1 (Id-1) has been implicated in tumor angiogenesis by regulating the expression of vascular endothelial growth factor (VEGF), but its molecular mechanism has not been fully understood. Here, we show the cross talk between Id-1 and hypoxia-inducible factor-1α (HIF-1α), that Id-1 induces VEGF by enhancing the stability and activity of HIF-1α in human endothelial and breast cancer cells. Although both the transcript and proteins levels of VEGF were induced by Id-1, only the protein expression of HIF-1α was induced without transcriptional changes in both human umbilical endothelial cells and MCF7 breast cancer cells. Such induction of the HIF-1α protein did not require de novo protein synthesis but was dependent on the active extracellular response kinase (ERK) pathway. In addition, stability of the HIF-1α protein was enhanced in part by the reduced association of the HIF-1α protein with von Hippel-Lindau protein in the presence of Id-1. Furthermore, Id-1 enhanced nuclear translocation and the transcriptional activity of HIF-1α. Transcriptional activation of HIF-1–dependent promoters was dependent on the active ERK pathway, and the association of HIF-1α protein with cyclic AMP-responsive element binding protein was enhanced by Id-1. Finally, Id-1 induced tube formation in human umbilical endothelial cells, which also required active ERK signaling. In conclusion, we provide the molecular mechanism of the cross talk between HIF-1α and Id-1, which may play a critical role in tumor angiogenesis. (Mol Cancer Res 2007;5(4):321–9)

Mel-18 Negatively Regulates INK4a/ARF-Independent Cell Cycle Progression via Akt Inactivation in Breast Cancer

Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

UTX inhibits EMT-induced breast CSC properties by epigenetic repression of EMT genes in cooperation with LSD1 and HDAC1.

The histone H3K27 demethylase, UTX, is a known component of the H3K4 methyltransferase MLL complex, but its functional association with H3K4 methylation in human cancers remains largely unknown. Here we demonstrate that UTX loss induces epithelial–mesenchymal transition (EMT)‐mediated breast cancer stem cell (CSC) properties by increasing the expression of the SNAIL, ZEB1 and ZEB2 EMT transcription factors (EMT‐TFs) and of the transcriptional repressor CDH1. UTX facilitates the epigenetic silencing of EMT‐TFs by inducing competition between MLL4 and the H3K4 demethylase LSD1. EMT‐TF promoters are occupied by c‐Myc and MLL4, and UTX recognizes these proteins, interrupting their transcriptional activation function. UTX decreases H3K4me2 and H3 acetylation at these promoters by forming a transcriptional repressive complex with LSD1, HDAC1 and DNMT1. Taken together, our findings indicate that UTX is a prominent tumour suppressor that functions as a negative regulator of EMT‐induced CSC‐like properties by epigenetically repressing EMT‐TFs.

Loss of Mel-18 enhances breast cancer stem cell activity and tumorigenicity through activating Notch signaling mediated by the Wnt/TCF pathway

Mel‐18 has been proposed as a negative regulator of Bmi‐1, a cancer stem cell (CSC) marker, but it is still unclear whether Mel‐18 is involved in CSC regulation. Here, we examined the effect of Mel‐18 on the stemness of human breast CSCs. In Mel‐18 small hairpin RNA (shRNA)‐transduced MCF‐7 cells, side population (SP) cells and breast CSC surface marker (CD44+/CD24–/ESA+)‐expressing cells, which imply a CSC population, were enriched. Moreover, the self‐renewal of CSCs was enhanced by Mel‐18 knockdown, as measured by the ability for tumorsphere formation in vitro and tumor‐initiating capacity in vivo. Similarly, Mel‐18 overexpression inhibited the number and self‐renewal activity of breast CSCs in SK‐BR‐3 cells. Furthermore, our data showed that Mel‐18 blockade up‐regulated the expression of the Wnt/TCF target Jagged‐1, a Notch ligand, and consequently activated the Notch pathway. Pharmacologic inhibition of the Notch and Wnt pathways abrogated Mel‐18 knockdown‐mediated tumorsphere formation ability. Taken together, our findings suggest that Mel‐18 is a novel negative regulator of breast CSCs that inhibits the stem cell population and in vitro and in vivo self‐renewal through the inactivation of Wnt‐mediated Notch signaling.—Won, H.‐Y., Lee, J.‐Y., Shin, D.‐H., Park, J.‐H., Nam, J.‐S., Kim, H.‐C., Kong, G. Loss of Mel‐18 enhances breast cancer stem cell activity and tumorigenicity through activating Notch signaling mediated by the Wnt/TCF pathway. FASEB J. 26, 5002–5013 (2012). www.fasebj.org

Roles and epigenetic regulation of epithelial–mesenchymal transition and its transcription factors in cancer initiation and progression

The epithelial–mesenchymal transition (EMT) is a crucial developmental process by which epithelial cells undergo a mesenchymal phenotypic change. During EMT, epigenetic mechanisms including DNA methylation and histone modifications are involved in the regulation of EMT-related genes. The epigenetic gene silencing of the epithelial marker E-cadherin has been well characterized. In particular, three major transcriptional repressors of E-cadherin, Snail, ZEB, and Twist families, also known as EMT-inducing transcription factors (EMT-TFs), play a crucial role in this process by cooperating with multiple epigenetic modifiers. Furthermore, recent studies have identified the novel epigenetic modifiers that control the expression of EMT-TFs, and these modifiers have emerged as critical regulators of cancer development and as novel therapeutic targets for human cancer. In this review, the diverse functions of EMT-TFs in cancer progression, the cooperative mechanisms of EMT-TFs with epigenetic modifiers, and epigenetic regulatory roles for the expression of EMT-TFs will be discussed.

CBX7 inhibits breast tumorigenicity through DKK-1-mediated suppression of the Wnt/β-catenin pathway

Polycomb protein chromobox homolog 7 (CBX7) is involved in several biologic processes including stem cell regulation and cancer development, but its roles in breast cancer remain unknown. Here, we demonstrate that CBX7 negatively regulates breast tumor initiation. CD44+/CD24‐/ESA+ breast stem‐like cells showed diminished CBX7 expression. Furthermore, small hairpin RNA‐mediated CBX7 knockdown in breast epithelial and cancer cells increased the CD44+/CD24‐/ESA+ cell population and reinforced in vitro self‐renewal and in vivo tumor‐initiating ability. Similarly, CBX7 overexpression repressed these effects. We also found that CBX7 inhibits the Wnt/α‐catenin/T cell factor pathway by enhancing the expression of Dickkopf‐1 (DKK‐1), a Wnt antagonist. In particular, CBX7 increased DKK‐1 transcription by cooperating with p300 acetyltransferase and subsequently enhancing the histone acetylation of the DKK‐1 promoter. Furthermore, pharmacologic inhibition of DKK‐1 in CBX7‐overexpressing cells showed recovery of Wnt signaling and consequent rescue of the CD44+/CD24‐/ESA+ cell population. Taken together, these findings indicate that CBX7‐mediated epigenetic induction of DKK‐1 is crucial for the inhibition of breast tumorigenicity, suggesting that CBX7 could be a potential tumor suppressor in human breast cancer. —Kim, H.–Y., Park, J.–H., Won, H.–Y., Lee, J.–Y., Kong, G., CBX7 inhibits breast tumorigenicity through DKK‐1‐mediated suppression of the Wnt/β‐catenin pathway. FASEB J. 29, 300–313 (2015). www.fasebj.org

MEL-18 loss mediates estrogen receptor–α downregulation and hormone independence

The polycomb protein MEL-18 has been proposed as a tumor suppressor in breast cancer; however, its functional relevance to the hormonal regulation of breast cancer remains unknown. Here, we demonstrated that MEL-18 loss contributes to the hormone-independent phenotype of breast cancer by modulating hormone receptor expression. In multiple breast cancer cohorts, MEL-18 was markedly downregulated in triple-negative breast cancer (TNBC). MEL-18 expression positively correlated with the expression of luminal markers, including estrogen receptor-α (ER-α, encoded by ESR1). MEL-18 loss was also associated with poor response to antihormonal therapy in ER-α-positive breast cancer. Furthermore, whereas MEL-18 loss in luminal breast cancer cells resulted in the downregulation of expression and activity of ER-α and the progesterone receptor (PR), MEL-18 overexpression restored ER-α expression in TNBC. Consistently, in vivo xenograft experiments demonstrated that MEL-18 loss induces estrogen-independent growth and tamoxifen resistance in luminal breast cancer, and that MEL-18 overexpression confers tamoxifen sensitivity in TNBC. MEL-18 suppressed SUMOylation of the ESR1 transactivators p53 and SP1, thereby driving ESR1 transcription. MEL-18 facilitated the deSUMOylation process by inhibiting BMI-1/RING1B-mediated ubiquitin-proteasomal degradation of SUMO1/sentrin-specific protease 1 (SENP1). These findings demonstrate that MEL-18 is a SUMO-dependent regulator of hormone receptors and suggest MEL-18 expression as a marker for determining the antihormonal therapy response in patients with breast cancer.