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RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation.

Morales M, Arenas EJ, Urosevic J, Guiu M, Fernández E, Planet E, Fenwick RB, Fernández-Ruiz S, Salvatella X, Reverter D, Carracedo A, Massagué J, Gomis RR - EMBO Mol Med (2014)

Bottom Line: In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease.We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma.Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme.

View Article: PubMed Central - PubMed

Affiliation: Oncology Program, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

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RARRES3 prevents breast cancer lung metastasisSchematic representation of in vivo experimental procedure to evaluate lung metastatic potential from the orthotopic site. The indicated cell lines (5 × 105 cells) were injected contralaterally into the fourth mammary fat pad of mice. Tumors reaching 300 mm3 were surgically removed. Seven days post-mastectomy, lung metastasis burden originated from size-matched tumors was quantified.(Left panel) Quantification of ex vivo bioluminescent signal at the lungs in each experimental group subjected to the tumor growth/resection scheme described in (A) at end point. n = 8 and n = 9 mice per group were used. Whiskers plots from min–max values were used. (Right panel) Representative bioluminescence images of in vivo and ex vivo lung colonization of the mice are shown.Representative human Vimentin IHC staining of whole lung sections to highlight metastatic tumor lesions from (B) are shown. Inset panels (4× magnification) reflect the size and multiple metastatic foci detected in the LM2-Mock group.LM2 cells (5 × 105) expressing an empty vector (LM2-Mock) or a RARRES3-expressing vector (LM2-RARRES3) were injected contralaterally into the fourth mammary fat pad of mice, and tumor growth was measured over time. n = 20 per group. Data are averages ± SEM.
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fig02: RARRES3 prevents breast cancer lung metastasisSchematic representation of in vivo experimental procedure to evaluate lung metastatic potential from the orthotopic site. The indicated cell lines (5 × 105 cells) were injected contralaterally into the fourth mammary fat pad of mice. Tumors reaching 300 mm3 were surgically removed. Seven days post-mastectomy, lung metastasis burden originated from size-matched tumors was quantified.(Left panel) Quantification of ex vivo bioluminescent signal at the lungs in each experimental group subjected to the tumor growth/resection scheme described in (A) at end point. n = 8 and n = 9 mice per group were used. Whiskers plots from min–max values were used. (Right panel) Representative bioluminescence images of in vivo and ex vivo lung colonization of the mice are shown.Representative human Vimentin IHC staining of whole lung sections to highlight metastatic tumor lesions from (B) are shown. Inset panels (4× magnification) reflect the size and multiple metastatic foci detected in the LM2-Mock group.LM2 cells (5 × 105) expressing an empty vector (LM2-Mock) or a RARRES3-expressing vector (LM2-RARRES3) were injected contralaterally into the fourth mammary fat pad of mice, and tumor growth was measured over time. n = 20 per group. Data are averages ± SEM.

Mentions: We studied the functional role of RARRES3 in experimental models of BC metastasis to lung. We used the metastatic BC cell line MDA-MB-231-LM2 (LM2), which was selected in vivo on the basis of a high capacity to colonize the lungs in mice, and the corresponding parental cell line MDA-MB-231, namely parental cells (Minn et al, 2005). LM2 cells showed a fivefold lower RARRES3 expression than their parental counterparts (Supplementary Fig S2A and B) and have been described to rapidly colonize the lungs when inoculated orthotopically in the mammary fat pad of immunodeficient mice (Padua et al, 2008). We examined how RARRES3 overexpression (Supplementary Fig S2A, B and C) modified the capacity of LM2 cells to colonize the lungs (Fig 2A, B and C). Of note, modulation of RARRES3 levels did not significantly alter the expression of any other LMS gene in parental or LM2 cell derivatives (Supplementary Fig S2A and B). In detail, Mock and RARRES3-overexpressing LM2 cells were injected into the mammary fat pad (MFP) of BALB/c Nude mice, and tumors were allowed to grow until they reach 300 mm3. The tumors were then surgically resected, and lung colonization was allowed to develop (Fig 2A). Seven days after mastectomy of the primary tumor, we assayed metastatic activity by bioluminescence imaging (BLI) of luciferase-transduced LM2 cells both in live animals and in the lungs ex vivo (Fig 2B). While six out of eight mice inoculated with LM2-Mock cells presented luciferase activity in the lungs, bioluminescence was detected in only two out of nine animals injected with LM2-RARRES3 cells (Fig 2B). Moreover, the amount of luciferase detected differed significantly, as shown in representative in vivo and ex vivo images of the lungs (Fig 2B). The resulting metastatic lesions showed positive staining for human Vimentin by immunohistochemistry (IHC), which specifically stains human MDA-MB-231 cells (Fig 2C). Several metastatic foci were observed throughout the lungs of mice bearing LM2-Mock tumors, while these were hardly observed in mice bearing LM2-RARRES3 tumors. Interestingly, RARRES3 expression did not provide any growth advantage to cells when implanted at the MFP, as tested in an independent experiment (Fig 2D), or in vitro (Supplementary Fig S3A). RARRES3-expressing tumors did not display any change in vascular permeability, measured as effusion of intravenously injected rhodamine-conjugated dextran into the tumor or changes in VEGF expression levels (Supplementary Fig S3B and C). In addition, RARRES3 expression in primary tumors did not lead to differences in the number of circulating tumor cells, as measured by relative levels of human GAPDH to murine B2M (Supplementary Fig S3D). This observation suggests that the early steps of metastasis, including tumor vascularization and intravasation, were not under the influence of RARRES3 expression.


RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation.

Morales M, Arenas EJ, Urosevic J, Guiu M, Fernández E, Planet E, Fenwick RB, Fernández-Ruiz S, Salvatella X, Reverter D, Carracedo A, Massagué J, Gomis RR - EMBO Mol Med (2014)

RARRES3 prevents breast cancer lung metastasisSchematic representation of in vivo experimental procedure to evaluate lung metastatic potential from the orthotopic site. The indicated cell lines (5 × 105 cells) were injected contralaterally into the fourth mammary fat pad of mice. Tumors reaching 300 mm3 were surgically removed. Seven days post-mastectomy, lung metastasis burden originated from size-matched tumors was quantified.(Left panel) Quantification of ex vivo bioluminescent signal at the lungs in each experimental group subjected to the tumor growth/resection scheme described in (A) at end point. n = 8 and n = 9 mice per group were used. Whiskers plots from min–max values were used. (Right panel) Representative bioluminescence images of in vivo and ex vivo lung colonization of the mice are shown.Representative human Vimentin IHC staining of whole lung sections to highlight metastatic tumor lesions from (B) are shown. Inset panels (4× magnification) reflect the size and multiple metastatic foci detected in the LM2-Mock group.LM2 cells (5 × 105) expressing an empty vector (LM2-Mock) or a RARRES3-expressing vector (LM2-RARRES3) were injected contralaterally into the fourth mammary fat pad of mice, and tumor growth was measured over time. n = 20 per group. Data are averages ± SEM.
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fig02: RARRES3 prevents breast cancer lung metastasisSchematic representation of in vivo experimental procedure to evaluate lung metastatic potential from the orthotopic site. The indicated cell lines (5 × 105 cells) were injected contralaterally into the fourth mammary fat pad of mice. Tumors reaching 300 mm3 were surgically removed. Seven days post-mastectomy, lung metastasis burden originated from size-matched tumors was quantified.(Left panel) Quantification of ex vivo bioluminescent signal at the lungs in each experimental group subjected to the tumor growth/resection scheme described in (A) at end point. n = 8 and n = 9 mice per group were used. Whiskers plots from min–max values were used. (Right panel) Representative bioluminescence images of in vivo and ex vivo lung colonization of the mice are shown.Representative human Vimentin IHC staining of whole lung sections to highlight metastatic tumor lesions from (B) are shown. Inset panels (4× magnification) reflect the size and multiple metastatic foci detected in the LM2-Mock group.LM2 cells (5 × 105) expressing an empty vector (LM2-Mock) or a RARRES3-expressing vector (LM2-RARRES3) were injected contralaterally into the fourth mammary fat pad of mice, and tumor growth was measured over time. n = 20 per group. Data are averages ± SEM.
Mentions: We studied the functional role of RARRES3 in experimental models of BC metastasis to lung. We used the metastatic BC cell line MDA-MB-231-LM2 (LM2), which was selected in vivo on the basis of a high capacity to colonize the lungs in mice, and the corresponding parental cell line MDA-MB-231, namely parental cells (Minn et al, 2005). LM2 cells showed a fivefold lower RARRES3 expression than their parental counterparts (Supplementary Fig S2A and B) and have been described to rapidly colonize the lungs when inoculated orthotopically in the mammary fat pad of immunodeficient mice (Padua et al, 2008). We examined how RARRES3 overexpression (Supplementary Fig S2A, B and C) modified the capacity of LM2 cells to colonize the lungs (Fig 2A, B and C). Of note, modulation of RARRES3 levels did not significantly alter the expression of any other LMS gene in parental or LM2 cell derivatives (Supplementary Fig S2A and B). In detail, Mock and RARRES3-overexpressing LM2 cells were injected into the mammary fat pad (MFP) of BALB/c Nude mice, and tumors were allowed to grow until they reach 300 mm3. The tumors were then surgically resected, and lung colonization was allowed to develop (Fig 2A). Seven days after mastectomy of the primary tumor, we assayed metastatic activity by bioluminescence imaging (BLI) of luciferase-transduced LM2 cells both in live animals and in the lungs ex vivo (Fig 2B). While six out of eight mice inoculated with LM2-Mock cells presented luciferase activity in the lungs, bioluminescence was detected in only two out of nine animals injected with LM2-RARRES3 cells (Fig 2B). Moreover, the amount of luciferase detected differed significantly, as shown in representative in vivo and ex vivo images of the lungs (Fig 2B). The resulting metastatic lesions showed positive staining for human Vimentin by immunohistochemistry (IHC), which specifically stains human MDA-MB-231 cells (Fig 2C). Several metastatic foci were observed throughout the lungs of mice bearing LM2-Mock tumors, while these were hardly observed in mice bearing LM2-RARRES3 tumors. Interestingly, RARRES3 expression did not provide any growth advantage to cells when implanted at the MFP, as tested in an independent experiment (Fig 2D), or in vitro (Supplementary Fig S3A). RARRES3-expressing tumors did not display any change in vascular permeability, measured as effusion of intravenously injected rhodamine-conjugated dextran into the tumor or changes in VEGF expression levels (Supplementary Fig S3B and C). In addition, RARRES3 expression in primary tumors did not lead to differences in the number of circulating tumor cells, as measured by relative levels of human GAPDH to murine B2M (Supplementary Fig S3D). This observation suggests that the early steps of metastasis, including tumor vascularization and intravasation, were not under the influence of RARRES3 expression.

Bottom Line: In estrogen receptor-negative breast cancer patients, metastatic relapse usually occurs in the lung and is responsible for the fatal outcome of the disease.We show that RARRES3 downregulation engages metastasis-initiating capabilities by facilitating adhesion of the tumor cells to the lung parenchyma.Our results establish RARRES3 downregulation as a potential biomarker to identify patients at high risk of lung metastasis who might benefit from a differentiation treatment in the adjuvant programme.

View Article: PubMed Central - PubMed

Affiliation: Oncology Program, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

Show MeSH
Related in: MedlinePlus