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Supplementary Information and Figures - PDF file 1406K, Figure S1 provides additional data on PDK1-induced oncogenic transformation; Figure S2, Synthetic Lethal Screening Identifies PLK1 as a Crucial Downstream Effector of PDK1 to Mediate Cancer Cell Survival; Figure S3 shows PLK1 Inhibition Decrease Myc Protein expression in various Cancer Cell Lines; Figures S4 shows Genetic and Pharmacologic Inhibition of PDK1 Blocks PLK1 Activity in Cancer Cells; Figure S5 shows PDK1 Drives Cancer Initiating Cell Maintenance and Self-Renewal; Figure S6 shows that PDK1-induced gene signature associated with human cancers and patient survival; Figure S7 shows BI2356 synergizes with BEZ235 to induce synthetic lethality in CRC both in vitro and in vivo; Figure S8 shows The Effects of BI2536 in Combination with PP242 or Rapamycin on Apoptosis and proliferation; Supplementary table 1 shows the expression profiles of PDK1-regulated genes in HEK-Vector, -PDK1, -Myc and -E545K cells

Table S2 - XLS file 203K, Supplementary table 2 shows the expression profiles of Myc-regulated genes in HEK-Vector, -PDK1, -Myc and -E545K cells

Table S6 - XLS file 22K, Supplementary table 6 shows primers used for quantitative RT-PCR analysis

Table S5 - XLS file 30K, Supplementary table 5 show the list of genes overlapped with published database

Table S4 - XLS file 55K, Supplementary table 4 shows the list of PDK1-regulated genes significantly different from Myc-regulated genes

Table S3 - XLS file 71K, Supplementary table 3 shows the expression profiles of PIK3CA/E545K-regulated genes in HEK-Vector, -PDK1, -Myc and -E545K cells

Table S1 - XLS file 316K, Supplementary table 1 shows the expression profiles of PDK1-regulated genes in HEK-Vector, -PDK1, -Myc and -E545K cells

Epithelial-mesenchymal transition (EMT) in cancer cells plays a pivotal role in determining metastatic prowess, but knowledge of EMT regulation remains incomplete. In this study, we defined a critical functional role for the Forkhead transcription factor FOXQ1 in regulating EMT in breast cancer cells. FOXQ1 expression was correlated with high-grade basal-like breast cancers and was associated with poor clinical outcomes. RNAi-mediated suppression of FOXQ1 expression in highly invasive human breast cancer cells reversed EMT, reduced invasive ability, and alleviated other aggressive cancer phenotypes manifested in 3-dimensional Matrigel (BD Biosciences) culture. Conversely, enforced expression of FOXQ1 in differentiated human mammary epithelial cells (HMLER) or epithelial cancer cell lines provoked an epithelial to mesenchymal morphologic change, gain of stem cell–like properties, and acquisition of resistance to chemotherapy-induced apoptosis. Mechanistic investigations revealed that FOXQ1-induced EMT was associated with transcriptional inactivation of the epithelial regulator E-cadherin (CDH1). Our findings define a key role for FOXQ1 in regulating EMT and aggressiveness in human cancer. Cancer Res; 71(8); 3076–86. ©2011 AACR.

Supplementary Table 2 from FOXQ1 Regulates Epithelial-Mesenchymal Transition in Human Cancers

Supplementary Table 3 from FOXQ1 Regulates Epithelial-Mesenchymal Transition in Human Cancers

Supplementary Table 1 from FOXQ1 Regulates Epithelial-Mesenchymal Transition in Human Cancers

Both EZH2 and NF-κB contribute to aggressive breast cancer, yet whether the two oncogenic factors have functional crosstalk in breast cancer is unknown. Here, we uncover an unexpected role of EZH2 in conferring the constitutive activation of NF-κB target gene expression in ER-negative basal-like breast cancer cells. This function of EZH2 is independent of its histone methyltransferase activity but requires the physical interaction with RelA/RelB to promote the expression of NF-κB targets. Intriguingly, EZH2 acts oppositely in ER-positive luminal-like breast cancer cells and represses NF-κB target gene expression by interacting with ER and directing repressive histone methylation on their promoters. Thus, EZH2 functions as a double-facet molecule in breast cancers, either as a transcriptional activator or repressor of NF-κB targets, depending on the cellular context. These findings reveal an additional mechanism by which EZH2 promotes breast cancer progression and underscore the need for developing context-specific strategy for therapeutic targeting of EZH2 in breast cancers.

Although 3-phosphoinositide–dependent protein kinase-1 (PDK1) has been predominately linked to the phosphoinositide 3-kinase (PI3K)–AKT pathway, it may also evoke additional signaling outputs to promote tumorigenesis. Here, we report that PDK1 directly induces phosphorylation of Polo-like kinase 1 (PLK1), which in turn induces MYC phosphorylation and protein accumulation. We show that PDK1–PLK1–MYC signaling is critical for cancer cell growth and survival, and small-molecule inhibition of PDK1/PLK1 provides an effective approach for therapeutic targeting of MYC dependency. Intriguingly, PDK1–PLK1–MYC signaling induces an embryonic stem cell–like gene signature associated with aggressive tumor behaviors and is a robust signaling axis driving cancer stem cell (CSC) self-renewal. Finally, we show that a PLK1 inhibitor synergizes with an mTOR inhibitor to induce synergistic antitumor effects in colorectal cancer by antagonizing compensatory MYC induction. These findings identify a novel pathway in human cancer and CSC activation and provide a therapeutic strategy for targeting MYC-associated tumorigenesis and therapeutic resistance. Significance: This work identifies PDK1–PLK1–MYC signaling as a new oncogenic pathway driving oncogenic transformation and CSC self-renewal. Targeted inhibition of PDK1/PLK1 is robust in targeting MYC dependency in cancer cells. Thus, our findings provide important insights into cancer and CSC biology and have significant therapeutic implications. Cancer Discov; 3(10); 1156–71. ©2013 AACR. See related commentary by Cunningham and Ruggero, p. 1099 This article is highlighted in the In This Issue feature, p. 1083

Epithelial-mesenchymal transition (EMT) in cancer cells plays a pivotal role in determining metastatic prowess, but knowledge of EMT regulation remains incomplete. In this study, we defined a critical functional role for the Forkhead transcription factor FOXQ1 in regulating EMT in breast cancer cells. FOXQ1 expression was correlated with high-grade basal-like breast cancers and was associated with poor clinical outcomes. RNAi-mediated suppression of FOXQ1 expression in highly invasive human breast cancer cells reversed EMT, reduced invasive ability, and alleviated other aggressive cancer phenotypes manifested in 3-dimensional Matrigel (BD Biosciences) culture. Conversely, enforced expression of FOXQ1 in differentiated human mammary epithelial cells (HMLER) or epithelial cancer cell lines provoked an epithelial to mesenchymal morphologic change, gain of stem cell–like properties, and acquisition of resistance to chemotherapy-induced apoptosis. Mechanistic investigations revealed that FOXQ1-induced EMT was associated with transcriptional inactivation of the epithelial regulator E-cadherin (CDH1). Our findings define a key role for FOXQ1 in regulating EMT and aggressiveness in human cancer. Cancer Res; 71(8); 3076–86. ©2011 AACR.

Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. We found that DZNep effectively depleted cellular levels of PRC2 components EZH2, SUZ12, and EED and inhibited associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi), genome-wide expression analysis, and chromatin immunoprecipitation (ChIP) studies, we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep. We further demonstrate that the preferential reactivation of a set of these genes by DZNep, including a novel apoptosis affector, FBXO32, contributes to DZNep-induced apoptosis in breast cancer cells. Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy.

Differential co-expression signifies the deregulated pathways as opposed to differential expression that signifies change of gene expression. Kostka and Spang proposed a score and an algorithm to elicit differentially co-expressed gene-sets. We analyze the statistical properties of their score in two different data processing settings and obtain respective null-distributions to provide the statistical significance of a gene-set through the p-value of its score. We propose to use these p-values to automate their algorithm. In addition, we propose a two stage algorithm, based on Friendly Neighbors (FNs) algorithm, called FNs-KS algorithm for improved discovery of such gene set i.e. improves both sensitivity and specificity of the discovery.

CDKN1C (encoding tumor suppressor p57KIP2) is a cyclin-dependent kinase (CDK) inhibitor whose family members are often transcriptionally downregulated in human cancer via promoter DNA methylation. In this study, we show that CDKN1C is repressed in breast cancer cells mainly through histone modifications. In particular, we show that CDKN1C is targeted by histone methyltransferase EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3), and can be strongly activated by inhibition of EZH2 in synergy with histone deacetylase inhibitor. Consistent with the overexpression of EZH2 in a variety of human cancers including breast cancer, CDKN1C in these cancers is downregulated, and breast tumors expressing low levels of CDKN1C are associated with a poor prognosis. We further show that assessing both EZH2 and CDKN1C expression levels as a measurement of EZH2 pathway activity provides a more predictive power of disease outcome than that achieved with EZH2 or CDKN1C alone. Taken together, our study reveals a novel epigenetic mechanism governing CDKN1C repression in breast cancer. Importantly, as a newly identified EZH2 target with prognostic value, it has implications in patient stratification for cancer therapeutic targeting EZH2-mediated gene repression.