Finding another transcription factor that regulates metastasis in mice using orthotopic animal model
It is the second paper I read recently using the "mouse sorter" approach. This one injected cells into mammary fat pad and looked for metastatic cells in lung.
Detailed method is
Functional screen in an orthotopic animal model and validation of metastasis-suppressing activity.
To establish the animal model for the forward genetic screen, 1 × 10E6 168FARN–Luc cells containing a genome-wide mouse RNAi library covering 40,000 genes (System Biosciences) were orthotopically transplanted into the mammary fat pads of 10-week-old BALB/c mice (National Cancer Institute). Five mice were used in the initial screen. Mice bearing luciferase-positive tumours were imaged 7–8 weeks after transplantation with the aid of IVIS 200 Imaging system (Xenogen Corporation). Lung metastasis nodules were isolated and the genomic DNA of lung metastatic cells was purified with the Genomic DNA Purification kit according to manufacturer’s instructions (Qiagen). PCRs were performed according to the manufacturer’s protocol (SBI). PCR products were cloned with a TA cloning kit (Invitrogen) and sequenced. To validate the hits from the screen, 168FARN–Luc cells were transduced with lentiviruses containing KLF17 shRNA or KLF17 shRNA and KLF17 cDNA, which lacks an shRNA binding site; 1 × 10E6 cells were transplanted into the mammary fat pads of mice and imaged 7–8 weeks later. MCF7 human breast cancer cells (7 × 10E6 cells per mouse) stably expressing KLF17 shRNA were transplanted into the mammary fat pads of female SCID mice (6–8 weeks old). A slow-release pellet of 17β-oestradiol (1.7 mg, 90‑day release; Innovative Research of America) was implanted subcutaneously in the dorsal interscapular region before the transplantation of MCF7 cells. [WHY? WHY? WHY?] Mice bearing luciferase-positive tumours were imaged 6‑12 weeks after transplantation with the aid of IVIS 200 Imaging system (Xenogen Corporation).
To analyse the effect of KLF17 overexpression on 4T1 cells, these cells were transduced with a lentivirus containing mouse KLF17 cDNA and transplanted as described above. 168FARN–Luc cells containing non-target shRNA as a control were also transplanted as described above. To determine the number of metastasis nodules on the surface of the lungs, mouse lungs were collected and fixed with Fekete’s solution (60% ethanol, 3% formaldehyde and 4% glacial acetic acid) after intratracheal injection of a 15% india ink solution as described previously 54. The total number of unstained nodules on the lung surface was counted 4–5 weeks after transplantation.
To analyse the effect of Id1 overexpression on 168FARN and MCF7 cells, 168FARN and MCF7 cells were transduced with a lentivirus containing mouse or human Id1 cDNA, respectively, and were transplanted into female Balb/c or SCID mice, respectively, as described above. Mice bearing luciferase-positive tumours were imaged with the aid of IVIS 200 Imaging system (Xenogen Corporation, Hopkinton) as described above.
To study the effect of downregulation of KLF17 expression and Ras in tumour metastasis, NmuMG cells stably expressing a control vector, KLF17 shRNA, oncogenic HRasG12V or co-expressing KLF17 shRNA and HRasG12V were transplanted into SCID mice, as described above. Mice bearing luciferase-positive tumours were imaged with the aid of Xenogen IVIS 200 Imaging system, as described above.
Abstract
Nat Cell Biol. 2009 Oct 4.
KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer.
Gumireddy K, Li A, Gimotty PA, Klein-Szanto AJ, Showe LC, Katsaros D, Coukos G, Zhang L, Huang Q.
The Wistar Institute, 3601 Spruce Street, PA 19104, USA.
Metastasis is a complex multistep process, which requires the concerted action of many genes and is the primary cause of cancer death. Both pathways that regulate metastasis enhancement and those that regulate its suppression contribute to the tumour dissemination process. To identify new metastasis suppressors, we set up a forward genetic screen in a mouse model. We transduced a genome-wide RNA interference (RNAi) library into the non-metastatic 168FARN breast cancer cell line and orthotopically transplanted the cells into mouse mammary fat pads. We then selected cells that could metastasize to the lung and identified an RNAi for the KLF17 gene. Conversely, we demonstrate that ectopic expression of KLF17 in a highly metastatic 4T1 breast cancer cell line inhibits the ability of cells to metastasize from the mammary fat pad to the lung. We also show that suppression of KLF17 expression promotes breast cancer cell invasion and epithelial-mesenchymal transition (EMT), and that KLF17 protein functions by directly binding to the promoter region of Id1 (which encodes a key metastasis regulator in breast cancer) to inhibit its transcription. Finally, we demonstrate that KLF17 expression is significantly downregulated in primary human breast cancer samples and that the combined expression pattern of KLF17 and Id1 can serve as a potential biomarker for lymph node metastasis in breast cancer.
Avri Ben-Ze'ev
Weizmann Institute of Science, IsraelI found this work interesting since it identified the transcription factor KLF17 as an inhibitor of an early step in breast cancer metastasis and studied some of the mechanisms of its function. It points to an approach that could be used for identifying suppressors of later stages in metastasis.
By introducing a genome-wide RNA interference (RNAi) library into a non-metastatic breast cancer cell line, the authors identified KLF17 as an inhibitor of tumor cell motility and invasion. They found that KLF17 promotes epithelial-mesenchymal transition (EMT) and operates by binding to the promoter of the Id1 gene. The downregulation of KLF17 was found to cooperate with Ras in promoting metastasis. A low level of KLF17 and high level of Id1 expression was also found to be a good predictor of breast cancer metastasis in human breast cancer tissue samples. Future studies on unraveling the signaling pathways that link between KLF17, Id1 and Ras in breast cancer metastasis will be of much importance.
Hervé Acloque and M. Angela Nieto
Instituto de Neurociencias de Alicante, SpainThis paper identifies a transcription factor, Krüppel-like factor 17 (KLF17), which blocks metastasis formation by repressing the inhibitor of differentiation 1 (Id1) factor previously involved in the process of epithelial to mesenchymal transition (EMT).
The EMT is a crucial biological process, where epithelial cells lose their typical properties and become mesenchymal. EMT confers cell motility, invasive, and stem cell-like properties. EMT is fundamental during embryonic development, but its aberrant activation can lead to various pathologies in the adult (reviewed in {1} in which both Hervé Acloque and I are listed as authors). Much effort has been devoted to the characterization of EMT inducers (reviewed in {2}), but we know very little about its negative regulators. In the present study, Gumireddy et al. used a very elegant screening, based on a short hairpin RNA (shRNA) lentiviral library, to decipher a specific step of cancer progression that allows tumor cells to escape from the primary tumor. They identified a new transcription factor, KLF17, able to block metastasis, probably by repressing the induction of EMT and the delamination from the primary tumour, as shown with in vitro data. KLF17 acts by directly repressing Id1 expression, a previously described EMT inducer and pro-metastatic modulator. Future work will clarify the relationship between KLF17 and the known EMT inducers such as Snail, Zeb (zinc finger homeodomain enhancer-binding protein) or Twist factors, splicing modulators, such as epithelial splicing regulatory proteins (ESRPs), and microRNAs.
References: {1} Acloque et al. J Clin Invest 2009, 119:1438-49 [PMID: 19487820]. {2} Peinado et al. Nat Rev Cancer 2007, 7:415-28 [PMID: 17508028].
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