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VEGF189 overexpression in breast cancer cells delays metastasis

ABSTRACT: Vascular endothelial growth factor is a multifunctional cytokine playing important roles in angiogenesis, tumor progression and metastasis. Alternative splicing results in the production of several different isoforms of VEGF. We have previously generated human breast cancer cells overexpressing VEGF165 or VEGF189 isoforms (referred to as the V165 and V189 clones, respectively) and showed that VEGF189-transfected cells were less tumorigenic. In this study, we used bioluminescence imaging to analyze the metastasis capacity of breast cancer cell lines (MDA-MB-321) overexpressing VEGF isoforms in nude mice. V165, V189 and control cV clones were transfected with a luciferase plasmid to generate bioluminescent clones (the V165-B, V189-B and cV clones, respectively). These clones were then injected into the left heart ventricle of nude mice. Analysis of the location of metastases shows that V189-B cells induced fewer metastases in lung and bone than V165-B and cV-B cells. Moreover, there was a delay on metastasis appearance in mice receiving VEGF V189-B clone, consistent with increase survival in these mice. We investigated the possible mechanisms underlying these effects and found that VEGF189 increased adhesion and decreased cell invasion and survival in vitro. In the other side, transcriptomic analyses shown expression of genes, implicated in breast cancer and metastatic dissemination, in VEGF165 overexpressing cells. After in silico analyses of genes differentially expressed between V189 and V165 cells,we used Q-PCR assays to quantify mRNA levels of some gene of interest in 120 breast tumors from patients with or without metastasis and/or specially lung metastasis. We found that genes have prognostical significance and /or showed an influence on relapse-free survival. These data provide the first evidence that different VEGF isoforms have different effects on breast cancer cell line invasion and colonization. Human MDA-MB-231 breast cancer cells were used to generate stable transfected clones overexpressing VEGF165 isoform, VEGF189 isoform, or control vector (referred to as 42ctl8, 13ctl3 and PCIctl3, respectively). Analysis of the metastasis capacity of these clones in nude mice.

ORGANISM(S): Homo sapiens

SUBMITTER: Di Benedetto M

PROVIDER: S-ECPF-GEOD-37911 | biostudies-other | 2015 Sep

REPOSITORIES: biostudies-other

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Project description:Vascular endothelial growth factor is a multifunctional cytokine playing important roles in angiogenesis, tumor progression and metastasis. Alternative splicing results in the production of several different isoforms of VEGF. We have previously generated human breast cancer cells overexpressing VEGF165 or VEGF189 isoforms (referred to as the V165 and V189 clones, respectively) and showed that VEGF189-transfected cells were less tumorigenic. In this study, we used bioluminescence imaging to analyze the metastasis capacity of breast cancer cell lines (MDA-MB-321) overexpressing VEGF isoforms in nude mice. V165, V189 and control cV clones were transfected with a luciferase plasmid to generate bioluminescent clones (the V165-B, V189-B and cV clones, respectively). These clones were then injected into the left heart ventricle of nude mice. Analysis of the location of metastases shows that V189-B cells induced fewer metastases in lung and bone than V165-B and cV-B cells. Moreover, there was a delay on metastasis appearance in mice receiving VEGF V189-B clone, consistent with increase survival in these mice. We investigated the possible mechanisms underlying these effects and found that VEGF189 increased adhesion and decreased cell invasion and survival in vitro. In the other side, transcriptomic analyses shown expression of genes, implicated in breast cancer and metastatic dissemination, in VEGF165 overexpressing cells. After in silico analyses of genes differentially expressed between V189 and V165 cells,we used Q-PCR assays to quantify mRNA levels of some gene of interest in 120 breast tumors from patients with or without metastasis and/or specially lung metastasis. We found that genes have prognostical significance and /or showed an influence on relapse-free survival. These data provide the first evidence that different VEGF isoforms have different effects on breast cancer cell line invasion and colonization. Human MDA-MB-231 breast cancer cells were used to generate stable transfected clones overexpressing VEGF165 isoform, VEGF189 isoform, or control vector (referred to as 42ctl8, 13ctl3 and PCIctl3, respectively). Analysis of the metastasis capacity of these clones in nude mice.

Project description:IntroductionCurrent studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.MethodIn the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.ResultsMUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-?, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.ConclusionsMUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.

Project description:Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering. SIGNIFICANCE: This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.

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VEGF189 overexpression in breast cancer cells delays metastasis

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