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TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

ABSTRACT: The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated the role of TRIM28 protein in the regulation of breast cancer stem cells (CSC) populations and tumorigenesis in vitro and in vivo. Downregulation of TRIM28 expression in xenografts led to deceased expression of pluripotency and mesenchymal markers, as well as inhibition of signaling pathways involved in the complex mechanism of CSC maintenance. Moreover, TRIM28 depletion reduced the ability of cancer cells to induce tumor growth when subcutaneously injected in limiting dilutions. Our data demonstrate that the downregulation of TRIM28 gene expression reduced the ability of CSCs to self-renew that resulted in significant reduction of tumor growth. Loss of function of TRIM28 leads to dysregulation of cell cycle, cellular response to stress, cancer cell metabolism, and inhibition of oxidative phosphorylation. All these mechanisms directly regulate maintenance of CSC population. Our original results revealed the role of the TRIM28 in regulating the CSC population in breast cancer. These findings may pave the way to novel and more effective therapies targeting cancer stem cells in breast tumors.

SUBMITTER: Czerwinska P

PROVIDER: S-EPMC5352203 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

Czerwińska Patrycja P Shah Parantu K PK Tomczak Katarzyna K Klimczak Marta M Mazurek Sylwia S Sozańska Barbara B Biecek Przemysław P Korski Konstanty K Filas Violetta V Mackiewicz Andrzej A Andersen Jannik N JN Wiznerowicz Maciej M

Oncotarget 20170101 1

The expression of Tripartite motif-containing protein 28 (TRIM28)/Krüppel-associated box (KRAB)-associated protein 1 (KAP1), is elevated in at least 14 tumor types, including solid and hematopoietic tumors. High level of TRIM28 is associated with triple-negative subtype of breast cancer (TNBC), which shows higher aggressiveness and lower survival rates. Interestingly, TRIM28 is essential for maintaining the pluripotent phenotype in embryonic stem cells. Following on that finding, we evaluated th ...[more]

PMID: 27845900

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Project description:KAP1 is overexpressed in breast cancer. To determine KAP1 regulated genes, we performed microarray analysis of gene expression in KAP1 depleted breast cancer cells MDA-MB-231LN. The transcriptional regulator TRIM28/KAP1 plays an important role in development, stem cell self-renewal, chromatin organization and the 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. Using the conserved zinc fingers linker region (ZnFL) as antigen, we have developed a ZnFL antibody that recognizes multiple KRAB-ZNFs. We showed that KAP1 and many KRAB-ZNFs were overexpressed in human breast cancers and breast cancer cell lines. In addition, an active SUMOylated form of KAP1 was markedly increased in breast cancer cells. Furthermore, KAP1 depletion in breast cancer cell lines reduced cell proliferation and inhibited tumor growth and metastasis of tumor xenografts. Conversely, KAP1 overexpression stimulated cell proliferation and tumor growth. KAP1 knockdown led to down-regulation of genes previously linked to tumor progression and metastasis, including PTGS2/COX2, EREG, CD44, MMP1 and MMP2. Interestingly, KAP1 depletion or genomic deletion led to dramatic down-regulation of multiple KRAB-ZNF proteins due in part to their increased degradation. KAP1-dependent stabilization of KRAB-ZNFs required a direct KRAB-ZNF-KAP1 interaction. These results establish KAP1 as a positive regulator of multiple KRAB-ZNFs and an important factor in the development of breast cancer. 7 total samples were analyzed. Stable sublines of MDA-MB-231LN cells expressing control non-targeting shRNA (Scr, 3 biological replicates) and two different shRNAs against KAP1 (KAP1-3, 2 biological replicates and KAP1-4, 2 biological replicates) from doxycycline-inducible pTRIPZ vector were cultured in the presence of 0.5 ug/ml doxycycline for 7 days to induce shRNA expression. Cells were lysed and total RNA was isolated using mirVana miRNA isolation kit (Ambion) in the WVU Genomics Core Facility.

Project description:TRIM28 emerged as a guard of the intrinsic "state of cell differentiation", facilitating self-renewal of pluripotent stem cells. Recent reports imply TRIM28 engagement in cancer stem cell (CSC) maintenance, although the exact mechanism remains unresolved. TRIM28 high expression is associated with worse melanoma patient outcomes. Here, we investigated the association between TRIM28 level and melanoma stemness, and aligned it with the antitumor immune response to find the mechanism of "stemness high/immune low" melanoma phenotype acquisition. Based on the SKCM TCGA data, the TRIM28 expression profile, clinicopathological features, expression of correlated genes, and the level of stemness and immune scores were analyzed in patient samples. The biological function for differentially expressed genes was annotated with GSEA. Results were validated with additional datasets from R2: Genomics Analysis and Visualization Platform and in vitro with a panel of seven melanoma cell lines. All statistical analyses were accomplished using GraphPad Prism 8. TRIM28HIGH-expressing melanoma patients are characterized by worse outcomes and significantly different gene expression profiles than the TRIM28NORM cohort. TRIM28 high level related to higher melanoma stemness as measured with several distinct scores and TRIM28HIGH-expressing melanoma cell lines possess the greater potential of melanosphere formation. Moreover, TRIM28HIGH melanoma tumors were significantly depleted with infiltrating immune cells, especially cytotoxic T cells, helper T cells, and B cells. Furthermore, TRIM28 emerged as a good predictor of "stemness high/immune low" melanoma phenotype. Our data indicate that TRIM28 might facilitate this phenotype by direct repression of interferon signaling. TRIM28 emerged as a direct link between stem cell-like phenotype and attenuated antitumor immune response in melanoma, although further studies are needed to evaluate the direct mechanism of TRIM28-mediated stem-like phenotype acquisition.

Dataset Information

TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

Publications

TRIM28 multi-domain protein regulates cancer stem cell population in breast tumor development.

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