KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer
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ABSTRACT: Metastasis is a complex multi-step process requiring the concerted action of many genes and is the primary cause of cancer deaths. Pathways that regulate metastasis enhancement and suppression both contribute to tumor dissemination process. In order to identify novel metastasis suppressors, we set up a forward genetic screen in a mouse model. We transduced a genome-wide RNAi library into the non-metastatic 168FARN breast cancer cell line, orthotopically transplanted the cells into mouse mammary fat pads, and then selected for cells that could metastasize to the lung and identified an RNAi for the KLF17 gene. Conversely, ectopic expression of KLF17 in highly metastatic 4T1 breast cancer cell line inhibited their ability to metastasize from the mammary fat pad to the lung. We showed that suppression of KLF17 expression promotes breast cancer cell invasion and epithelial-mesenchymal transition (EMT). We also showed that KLF17 functions by directly binding to the promoter of Id-1, a key metastasis regulator in breast cancer, to inhibit its transcription. Finally, we demonstrated that KLF17 expression is significantly down-regulated in primary human breast cancer samples and that the combined expression patterns of KLF17 and Id-1 can serve as a potential biomarker for lymph node metastasis in breast cancer.
Project description:Younger age and obesity increase the incidence and rates of metastasis of triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer. The tissue microenvironment, specifically the extracellular matrix (ECM), is known to promote tumor invasion and metastasis. We sought to characterize the effect of both age and obesity on the ECM of mammary fat pads. We used a diet-induced obesity (DIO) model where 10-week-old female mice were fed a high-fat diet (HFD) for 16 weeks or a control chow diet (CD) where time points were every 4 weeks to monitor age and obesity HFD progression. We isolated the mammary fat pads to characterize the ECM at each time point. Utilizing proteomics, we found that the early stages of obesity were sufficient to induce distinct differences in the ECM composition of mammary fat pads that promote TNBC cell invasion. ECM proteins previously implicated in driving TNBC invasion Collagen IV and Collagen VI, were enriched with weight gain. Together these data implicate ECM changes in the primary tumor microenvironment as mechanisms by which age and obesity contribute to breast cancer progression.
Project description:High Arid4b promotes mammary tumor growth and metastasis in mouse model systems, and is associated with poor metastasis-free survival in human breast cancer patients. Through shRNA-mediated knockdown, we demonstrated that loss of Arid4b significantly inhibits the ability of mouse breast cancer cells to metastasize to the lungs. We performed microarray expression and subsequent network analysis to identify genes diferentially regulated as a consequence of Arid4b knockdown.
Project description:High Arid4b promotes mammary tumor growth and metastasis in mouse model systems, and is associated with poor metastasis-free survival in human breast cancer patients. Through shRNA-mediated knockdown, we demonstrated that loss of Arid4b significantly inhibits the ability of mouse breast cancer cells to metastasize to the lungs. We performed microarray expression and subsequent network analysis to identify genes diferentially regulated as a consequence of Arid4b knockdown. The highly metastatic mouse breast cancer cell line 6DT1 was transduced with lentiviral shRNAs targeting Arid4b (RMM4534-NM_194262, Open Biosystems) or scrambled control in the same pLKO.1 vector backbone. Stably transduced cells were selected with puromycin, then total RNA was isolated from pooled clones.
Project description:MMTV-NeuNT transgenic mouse model harbors an activated form of Neu (NeuNT). Mice develop stochastically multifocal mammary adenocarcinomas that metastasize to the lung (Muller et al., 1988). MMTV-NeuNT mouse model exhibits both intravascular and parenchymal metastasis which provides a good tool to comprehensively study breast cancer metastasis. In this study, we investigated the role of TNC in tumor progression using the MMTV-NeuNT mouse model. (3 MMTV-NeuNT TNC WT v/s 3 MMTV-NeuNT TNC KO). Breast tumor tissue were collected 3 months after first tumor palpation.
Project description:It was demonstrated that mice with eIF4E that cannot be activated by phosphorylation (S209A) produced extracellular matrix (ECM) less conducive to breast cancer tumor invasion and metastasis. To examine whether this could in part bedue to differences in the protein composition we analyzed the proteome of soluble and insoluble ECM fractions derived from the mammary fat pad of mice with either WT or constitutively inactivated (S209A or "KI") eIF4E.
Project description:By combining RNAi-mediated knockdown of Id1 and Id3 in an aggressive mouse breast cancer cell line (4T1 cells) with genome-wide expression profiling, we identified several new Id target genes and novel pathways regulated by Id.
Project description:Breast cancer molecular subtypes preferentially metastasize to specific organs and the anatomical location of the metastasis is associated with the length of survival post-recurrence. We used microarrays to provide a detailed characterization of breast cancer site-specific metastases with particular focus on identifying genes predictive of breast cancer liver metastatic proprnsity
Project description:Our purpose is to identify candidate genes involved in the early steps of breast cancer metastasis and examine their pro-invasive functions both in vitro and in vivo. A percentage of bilateral breast cancers were clonally related based on copy number variation profiling. Whole exome sequencing and comparative sequence analysis revealed that a limited number of somatic mutations were acquired in this “breast to breast” metastasis. These mutations might promote breast cancer distant spread. The pro-invasive functions of a candidate metastasis gene were assessed in vitro by its abilities to promote proliferation, migration and invasion and in vivo as tumor xenografts in immunocompromised mice or a syngeneic orthotopic mouse breast cancer model. RNAseq analysis was performed to probe the transcription programs modulated by this candidate metastasis gene. SIVA1-D160N was one somatic mutation acquired in the breast to breast metastasis. Over-expression of SIVA1-D160N promoted migration and invasion of human MB-MDA-231 breast cancer cells in vitro, consistent with a dominant negative interfering function. When introduced via tail vein injection, 231 cells over-expressing SIVA1-D160N displayed enhanced distant spread on IVIS imaging. Over-expression of SIVA1-D160N promoted anchorage independent growth of mouse 4T1 breast cancer cells in vitro. When introduced orthotopically via mammary fat pad injection in syngeneic Balb/c mice, over-expression of SIVA1-D160N in 4T1 cells increased mammary gland tumor growth as well as liver metastasis. We conclude clonally related bilateral breast cancers represent a novel system to investigate metastasis and revealed a role of SIVA1-D160N in breast cancer metastasis.
Project description:Triple negative breast cancer cell line SUM159 labeled with triple reporter (TR, TK-GFP-fLUC) injected into Nude mice via mammary fat pad and tail vein injection. Tumor progression is monitored by in vivo bioluminescent imaging. Tumors from mammary and lung were isolated and cultured. These derivatives cells are further tested for their capability to develop mammary primary tumor and metastasis in lung and bone in mice. The total RNA samples are extracted from the panel of SUM159 and derivatives and the transcriptome profiles are determined by microarray. The correlation of transcriptome of the cells to metastasis capability indicated candidate genes and regulatory pathways of metastasis of TNBC.
Project description:Expression data from 4T1 subclones derived from mammary fat pad tumors (MFP), axillary lymph node tumors (AxLN), and axillary lymph node-derived lung metastases (AxLN-LuM). In parallel, expression data, in the same subclones, of tail vein-derived (TV) lung metastases. The mechanism of how lymph node metastases seed distant metastases is unknown. We used the 4T1 breast cancer cell line, which is an immune competent model of triple negative breast cancer and spontaneously metastasizes in balb/c mice. 4T1-GFP/fLuc cells were injected into MFP to form tumors and 4T1-mCherry/rLuc cells were injected into axillary lymph nodes to form tumors and then allowed to metastasize to lung. TV cells were allowed to metastasize in the lung. Cells were harvested at different time intervals after the injection. Tumors were extracted, dissociated, and then expanded in vitro to obtain MFP, AxLN, AxLN-LuM and TV-LuM subclones isolated after different time lags with respect to the injection.