Joseph B. Addison

Suppression of the Epithelial–Mesenchymal Transition by Grainyhead-like-2

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound-healing processes and the cancer stem cell-like compartment of tumors, including TGF-β dependence, we investigated the role of the Grainyhead gene, Grainyhead-like-2 (GRHL2) in oncogenic EMT. GRHL2 was downregulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir-200b/c as well as the TGF-β receptor antagonist, BMP2. Finally, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered an MET and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells.

A KLF4–miRNA-206 Autoregulatory Feedback Loop Can Promote or Inhibit Protein Translation Depending upon Cell Context

ABSTRACT Krüppel-like factor 4 (KLF4), a transcription factor that regulates cell fate in a context-dependent fashion, is normally induced upon growth arrest or differentiation. In many cancer cells there is dysregulation, with increased expression in proliferating cells. To identify sequence elements that mediate KLF4 suppression in normal epithelial cells, we utilized a luciferase reporter and RK3E cells, which undergo a proliferation-differentiation switch to form an epithelial sheet. A translational control element (TCE) within the KLF4 3′-untranslated region interacted with microRNAs (miRs) 206 and 344-1 to promote or inhibit KLF4 expression, respectively, in proliferating epithelial cells. Overall, the TCE suppressed expression in proliferating primary human mammary epithelial cells, but this suppressive effect was attenuated in immortalized mammary epithelial MCF10A cells, in which Dicer1 and miR-206 promoted KLF4 expression and TCE reporter activity. In contrast to MCF10A cells, in breast cancer cells the activity of miR-206 was switched, and it repressed KLF4 expression and TCE reporter activity. As miR-206 levels were KLF4 dependent, the results identify a KLF4–miR-206 feedback pathway that oppositely affects protein translation in normal cells and cancer cells. In addition, the results indicate that two distinct miRs can have opposite and competing effects on translation in proliferating cells.

The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells

Lung cancer remains the leading cause of cancer-related mortality for both men and women. Tumor recurrence and metastasis is the major cause of lung cancer treatment failure and death. The microRNA-200 (miR-200) family is a powerful regulator of the epithelial-mesenchymal transition (EMT) process, which is essential in tumor metastasis. Nevertheless, miR-200 family target genes that promote metastasis in non-small cell lung cancer (NSCLC) remain largely unknown. Here, we sought to investigate whether the microRNA-200 family regulates our previously identified NSCLC prognostic marker genes associated with metastasis, as potential molecular targets. Novel miRNA targets were predicted using bioinformatics tools based on correlation analyses of miRNA and mRNA expression in 57 squamous cell lung cancer tumor samples. The predicted target genes were validated with quantitative RT-PCR assays and western blot analysis following re-expression of miR-200a, -200b and -200c in the metastatic NSCLC H1299 cell line. The results show that restoring miR-200a or miR-200c in H1299 cells induces downregulation of DLC1, ATRX and HFE. Reinforced miR-200b expression results in downregulation of DLC1, HNRNPA3 and HFE. Additionally, miR-200 family downregulates HNRNPR3, HFE and ATRX in BEAS-2B immortalized lung epithelial cells in quantitative RT-PCR and western blot assays. The miR-200 family and these potential targets are functionally involved in canonical pathways of immune response, molecular mechanisms of cancer, metastasis signaling, cell-cell communication, proliferation and DNA repair in Ingenuity pathway analysis (IPA). These results indicate that re-expression of miR-200 downregulates our previously identified NSCLC prognostic biomarkers in metastatic NSCLC cells. These results provide new insights into miR-200 regulation in lung cancer metastasis and consequent clinical outcome, and may provide a potential basis for innovative therapeutic approaches for the treatment of this deadly disease.

KAP1 Promotes Proliferation and Metastatic Progression of Breast Cancer Cells

KAP1 (TRIM28) is a transcriptional regulator in embryonic development that controls stem cell self-renewal, chromatin organization, and the DNA damage response, acting as an essential corepressor for KRAB family zinc finger proteins (KRAB-ZNF). To gain insight into the function of this large gene family, we developed an antibody that recognizes the conserved zinc fingers linker region (ZnFL) in multiple KRAB-ZNF. Here, we report that the expression of many KRAB-ZNF along with active SUMOlyated KAP1 is elevated widely in human breast cancers. KAP1 silencing in breast cancer cells reduced proliferation and inhibited the growth and metastasis of tumor xenografts. Conversely, KAP1 overexpression stimulated cell proliferation and tumor growth. In cells where KAP1 was silenced, we identified multiple downregulated genes linked to tumor progression and metastasis, including EREG/epiregulin, PTGS2/COX2, MMP1, MMP2, and CD44, along with downregulation of multiple KRAB-ZNF proteins. KAP1-dependent stabilization of KRAB-ZNF required direct interactions with KAP1. Together, our results show that KAP1-mediated stimulation of multiple KRAB-ZNF contributes to the growth and metastasis of breast cancer.

Abstract LB-2: TRIM28 is overexpressed in breast cancer and regulates the expression of multiple cancer-associated genes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The transcriptional regulator TRIM28 plays an important role in development, stem cells self-renewal, chromatin organization and DNA damage response. KAP1 is an essential co-repressor for KRAB zinc finger proteins (KRAB-ZNFs). Though KRAB-ZNFs represent the largest family of human transcription factors, their biological functions are largely unknown. We showed that KAP1 and certain KRAB-ZNFs were overexpressed in breast tumors and breast cancer cell lines at both mRNA and protein levels. More significantly, an active SUMOylated form of KAP1 was markedly increased in breast cancer cells. KAP1 depletion in several breast cancer cell lines slowed cell proliferation and inhibited primary tumor growth and metastasis of MDA-MB-231LN xenografts, while KAP1 overexpression conversely stimulated cell proliferation and tumor growth. KAP1 knockdown led to down-regulation of PTGS2/COX2, EREG, CD44, MMP1, MMP2 and ID transcription factors, previously implicated in cancer progression and metastasis. These results indicate that KAP1 and KRAB-ZNFs are up-regulated in breast cancer cells and contribute to increased cell proliferation, tumor growth and metastasis. Citation Format: Joseph Addison, Colton Koontz, Sarah McLaughlin, Ryan Livengood, Elena Pugacheva, Dongquan Chen, Chad Creighton, Alexey V. Ivanov. TRIM28 is overexpressed in breast cancer and regulates the expression of multiple cancer-associated genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-2. doi:10.1158/1538-7445.AM2014-LB-2

Abstract B94: Gene expression profiling during EMT and MET identifies multiple transcription factors involved in the EMT transcriptional reprogramming

Background and Objective: The Epithelial-to-Mesenchymal Transition (EMT) is a developmental program which is often reactivated in aggressive metastatic breast cancer. Several transcription factors (TFs), such as Snail and ZEB can act as master regulators of EMT, directly repressing the gene for E-cadherin. We sought to identify common gene expression signatures characteristic to both EMT and a reverse process, Mesenchymal-to-Epithelial Transition (MET). Methods: Using Affymetrix Exon microarrays we characterized global gene expression changes in two cellular models: normal mammary epithelial HMLE cells induced to undergo EMT by expression of Snail1, Snail2 or ZEB1; and MDA-MB-231LN breast cancer cells induced to undergo MET by depletion of ZEB1 and ZEB2. Results: We identified two large gene sets: 1) 1017 genes down-regulated during EMT and up-regulated during MET, which we designated as MET-specific; and 2) 438 genes up-regulated during EMT and down-regulated during MET, which we designated as EMT-specific. 14 TFs were found in the EMT category. Their functional validation showed that individual overexpression of several of these TFs in HMLE cells was sufficient to elicit partial EMT. Discussion and Conclusions: Our comprehensive analysis of gene expression signatures common to EMT and MET identifies the EMT transcriptional program characteristic to invasive metastatic breast cancer. Grant support: NIH NCRR grant P20 RR16440 and Susan G. Komen for the Cure grant KG110350. Citation Format: Maria Voronkova, Joseph Addison, Renata Galeeva, James Denvir, Dongquan Chen, Alexey Ivanov. Gene expression profiling during EMT and MET identifies multiple transcription factors involved in the EMT transcriptional reprogramming. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B94.

Abstract 3125: KAP1 promotes malignant properties of breast cancer cells and regulates the expression of multiple KRAB-ZNFs.

Background and Objective: Chromatin remodeling is fundamental for normal development and is universally affected in cancer cells. The largest family of human transcription factors, KRAB-ZNF repressors, and their essential cofactor KAP1 play important roles in chromatin organization and regulate multiple cellular processes such as cell differentiation, pluripotency, apoptosis, DNA repair and imprinting. Here, we explored the role of KAP1 and KRAB-ZNFs in breast cancer. Methods: Using cancer patient samples, in vitro and animal xenograft model we analyzed KAP1 role in cancer cell proliferation and primary tumor formation. Results: Patient sample analysis showed KAP1 and KRAB-ZNFs overexpression in significant proportion of breast tumors. KAP1 depletion in a panel of breast cancer cell lines inhibited cancer cell proliferation, while KAP1 overexpression stimulated cell growth. Concordantly, KAP1 knockdown in MDA-MB-231LN human breast cancer cells inhibited primary tumor growth in orthotopic xenograft mouse model, while KAP1 overexpression had an opposite, growth stimulatory effect. KAP1 depletion resulted in decreased expression of a number of cancer-associated genes. Functional in vitro studies provided evidence that KAP1 positively regulates expression of multiple KRAB-ZNFs. Discussion and Conclusions: These results thus establish KAP1 as an oncogenic factor in breast cancer. Grant support: NIH NCRR grant P20 RR16440 and Susan G. Komen for the Cure grant KG110350. Citation Format: Joseph B. Addison, Renata Galeeva, Dongquan Chen, Elena N. Pugacheva, Alexey V. Ivanov. KAP1 promotes malignant properties of breast cancer cells and regulates the expression of multiple KRAB-ZNFs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3125. doi:10.1158/1538-7445.AM2013-3125

Abstract 3436: Suppression of the epithelial-mesenchymal transition (EMT) by a wound-healing gene, Grainyhead-like-2

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound healing processes and the cancer stem cell-like compartment of tumors, including TGF-β-dependence, we investigated the role of a Grainyhead gene (GRHL2) in oncogenic EMT. Grainyhead was specifically down-regulated in the claudin-low subclass of mammary tumors and in the basal-B subclass of breast cancer cell lines. Functionally, GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis-sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by its suppression of ZEB1 expression, by direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription, and up-regulated mir200b/c as well as the TGF-β receptor antagonist, BMP2. The expression of GRHL2 in the breast cancer cell line MDA-MB-231 triggered a mesenchymal-to-epithelial transition and sensitized the cells to anoikis. These results indicate that GRHL2 is a suppressor of the oncogenic EMT, and provide a novel molecular focus for the wound-healing/oncogenic EMT relationship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3436. doi:1538-7445.AM2012-3436