Limits...
Cripto-1 as a novel therapeutic target for triple negative breast cancer.

Castro NP, Fedorova-Abrams ND, Merchant AS, Rangel MC, Nagaoka T, Karasawa H, Klauzinska M, Hewitt SM, Biswas K, Sharan SK, Salomon DS - Oncotarget (2015)

Bottom Line: The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively.Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis.Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

View Article: PubMed Central - PubMed

Affiliation: Tumor Growth Factor Section, Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD, USA.

ABSTRACT
Triple-negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes and no current standard therapy. Here, we performed an in-depth molecular analysis of a mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resembled the triple-negative breast cancer (TNBC) both phenotypically and molecularly. Morphologically, primary tumors presented both epithelial and spindle-like cells but displayed only adenocarcinoma-like features in lung parenchyma. The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively. Cripto-1, an embryonic stem cell marker, was present in spindle-like areas and its promoter showed activity in primary tumors. Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis. Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

No MeSH data available.


Related in: MedlinePlus

Mouse Cripto-1 promoter and Cripto-1 knockout in JygMC(A) cell lineRepresentation of bioluminescent imaging on animals injected bilaterally into the fourth mammary gland with JygMC(A) cells containing the mouse Cripto-1 promoter. Animals were imaged at day 8, 16, 19 and 23-post cell injection. B.In situ detection of Cripto-1 promoter activity in primary tumor tissue sections. C. H&E of the primary tumor tissue sections depicted in B. showing carcinoma areas (green) and EMT-like areas (yellow) and high magnification (20X). Scale bars: 3mm and 200μm. D. Proliferation assay. JygMC(A) cells were seeded in 12-well dishes in triplicate at 5x104cells/well and cultured for 24, 48 and 72 hrs. Cells were then harvested and counted. Data are representative of two independent experiments in triplicate ±SD, *P < 0.0002, as compared to control cells. E. Average of primary tumor volumes are represented in the graph on JygCr-1KO and JygMC(A) parental cells (n = 7 animals/group ±SD, *P < 0.01 and **P < 0.0001, as compared to control animals). F. Number of pulmonary nodules per animal in JygCr-1KO and JygMC(A) parental animals (*P < 0.05, one-sided values; Wilcoxon rank-sum test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494913&req=5

Figure 7: Mouse Cripto-1 promoter and Cripto-1 knockout in JygMC(A) cell lineRepresentation of bioluminescent imaging on animals injected bilaterally into the fourth mammary gland with JygMC(A) cells containing the mouse Cripto-1 promoter. Animals were imaged at day 8, 16, 19 and 23-post cell injection. B.In situ detection of Cripto-1 promoter activity in primary tumor tissue sections. C. H&E of the primary tumor tissue sections depicted in B. showing carcinoma areas (green) and EMT-like areas (yellow) and high magnification (20X). Scale bars: 3mm and 200μm. D. Proliferation assay. JygMC(A) cells were seeded in 12-well dishes in triplicate at 5x104cells/well and cultured for 24, 48 and 72 hrs. Cells were then harvested and counted. Data are representative of two independent experiments in triplicate ±SD, *P < 0.0002, as compared to control cells. E. Average of primary tumor volumes are represented in the graph on JygCr-1KO and JygMC(A) parental cells (n = 7 animals/group ±SD, *P < 0.01 and **P < 0.0001, as compared to control animals). F. Number of pulmonary nodules per animal in JygCr-1KO and JygMC(A) parental animals (*P < 0.05, one-sided values; Wilcoxon rank-sum test).

Mentions: Since CRIPTO-1 physically interacts with all four Notch receptors and Notch4 and Cripto-1 has been shown to be relevant in embryogenesis, maintenance of a human BC stem cell population and tumorigenesis [6, 13, 14], we decided to investigate the role of Cripto-1 in the JygMC(A) mouse model. We generated a reporter system using the luciferase gene driven by the promoter for the mouse Cripto-1 gene. The mouse Cripto-1 promoter sequence can be found in Table S3. The reporter can detect real-time Cripto-1 expression in vitro and in vivo during tumor growth and lung metastasis. To validate the activity of the mouse Cripto-1 luciferase reporter in vitro, we performed a dual-luciferase assay using mouse F9 embryonal carcinoma cells that express high levels of endogenous Cripto-1 [37]. To induce the Cripto-1 promoter activity, we used two TGF-β-related family members: TGF-β1 and NODAL which were previously reported to activate the human Cripto-1 promoter [37] (Figure S4A and S4B). After construct validation, stably transfected JygMC(A) cells containing the mouse Cripto-1 promoter reporter construct or the negative-control were injected bilaterally into the fourth mammary fat pads (n = 5 animals/group). Tumor growth was assessed twice a week for the Cripto-1 promoter activity and weekly for negative-control animals. Using this unique detection system, we determined that the Cripto-1 promoter was expressed in 100% (10/10) of the injection sites of primary JygMC(A) mammary tumors (Figure 7A), whereas the same promoter construct was silenced in pulmonary metastases. This confirmed that this embryonic stem cell gene is only active during primary tumor growth and during the initiation of metastasis.


Cripto-1 as a novel therapeutic target for triple negative breast cancer.

Castro NP, Fedorova-Abrams ND, Merchant AS, Rangel MC, Nagaoka T, Karasawa H, Klauzinska M, Hewitt SM, Biswas K, Sharan SK, Salomon DS - Oncotarget (2015)

Mouse Cripto-1 promoter and Cripto-1 knockout in JygMC(A) cell lineRepresentation of bioluminescent imaging on animals injected bilaterally into the fourth mammary gland with JygMC(A) cells containing the mouse Cripto-1 promoter. Animals were imaged at day 8, 16, 19 and 23-post cell injection. B.In situ detection of Cripto-1 promoter activity in primary tumor tissue sections. C. H&E of the primary tumor tissue sections depicted in B. showing carcinoma areas (green) and EMT-like areas (yellow) and high magnification (20X). Scale bars: 3mm and 200μm. D. Proliferation assay. JygMC(A) cells were seeded in 12-well dishes in triplicate at 5x104cells/well and cultured for 24, 48 and 72 hrs. Cells were then harvested and counted. Data are representative of two independent experiments in triplicate ±SD, *P < 0.0002, as compared to control cells. E. Average of primary tumor volumes are represented in the graph on JygCr-1KO and JygMC(A) parental cells (n = 7 animals/group ±SD, *P < 0.01 and **P < 0.0001, as compared to control animals). F. Number of pulmonary nodules per animal in JygCr-1KO and JygMC(A) parental animals (*P < 0.05, one-sided values; Wilcoxon rank-sum test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494913&req=5

Figure 7: Mouse Cripto-1 promoter and Cripto-1 knockout in JygMC(A) cell lineRepresentation of bioluminescent imaging on animals injected bilaterally into the fourth mammary gland with JygMC(A) cells containing the mouse Cripto-1 promoter. Animals were imaged at day 8, 16, 19 and 23-post cell injection. B.In situ detection of Cripto-1 promoter activity in primary tumor tissue sections. C. H&E of the primary tumor tissue sections depicted in B. showing carcinoma areas (green) and EMT-like areas (yellow) and high magnification (20X). Scale bars: 3mm and 200μm. D. Proliferation assay. JygMC(A) cells were seeded in 12-well dishes in triplicate at 5x104cells/well and cultured for 24, 48 and 72 hrs. Cells were then harvested and counted. Data are representative of two independent experiments in triplicate ±SD, *P < 0.0002, as compared to control cells. E. Average of primary tumor volumes are represented in the graph on JygCr-1KO and JygMC(A) parental cells (n = 7 animals/group ±SD, *P < 0.01 and **P < 0.0001, as compared to control animals). F. Number of pulmonary nodules per animal in JygCr-1KO and JygMC(A) parental animals (*P < 0.05, one-sided values; Wilcoxon rank-sum test).
Mentions: Since CRIPTO-1 physically interacts with all four Notch receptors and Notch4 and Cripto-1 has been shown to be relevant in embryogenesis, maintenance of a human BC stem cell population and tumorigenesis [6, 13, 14], we decided to investigate the role of Cripto-1 in the JygMC(A) mouse model. We generated a reporter system using the luciferase gene driven by the promoter for the mouse Cripto-1 gene. The mouse Cripto-1 promoter sequence can be found in Table S3. The reporter can detect real-time Cripto-1 expression in vitro and in vivo during tumor growth and lung metastasis. To validate the activity of the mouse Cripto-1 luciferase reporter in vitro, we performed a dual-luciferase assay using mouse F9 embryonal carcinoma cells that express high levels of endogenous Cripto-1 [37]. To induce the Cripto-1 promoter activity, we used two TGF-β-related family members: TGF-β1 and NODAL which were previously reported to activate the human Cripto-1 promoter [37] (Figure S4A and S4B). After construct validation, stably transfected JygMC(A) cells containing the mouse Cripto-1 promoter reporter construct or the negative-control were injected bilaterally into the fourth mammary fat pads (n = 5 animals/group). Tumor growth was assessed twice a week for the Cripto-1 promoter activity and weekly for negative-control animals. Using this unique detection system, we determined that the Cripto-1 promoter was expressed in 100% (10/10) of the injection sites of primary JygMC(A) mammary tumors (Figure 7A), whereas the same promoter construct was silenced in pulmonary metastases. This confirmed that this embryonic stem cell gene is only active during primary tumor growth and during the initiation of metastasis.

Bottom Line: The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively.Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis.Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

View Article: PubMed Central - PubMed

Affiliation: Tumor Growth Factor Section, Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD, USA.

ABSTRACT
Triple-negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes and no current standard therapy. Here, we performed an in-depth molecular analysis of a mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resembled the triple-negative breast cancer (TNBC) both phenotypically and molecularly. Morphologically, primary tumors presented both epithelial and spindle-like cells but displayed only adenocarcinoma-like features in lung parenchyma. The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively. Cripto-1, an embryonic stem cell marker, was present in spindle-like areas and its promoter showed activity in primary tumors. Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis. Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

No MeSH data available.


Related in: MedlinePlus