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Dermcidin exerts its oncogenic effects in breast cancer via modulation of ERBB signaling.

Bancovik J, Moreira DF, Carrasco D, Yao J, Porter D, Moura R, Camargo A, Fontes-Oliveira CC, Malpartida MG, Carambula S, Vannier E, Strauss BE, Wakamatsu A, Alves VA, Logullo AF, Soares FA, Polyak K, Belizário JE - BMC Cancer (2015)

Bottom Line: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines.We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice.Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Institute of Biomedical Sciences - University of São Paulo, Av Lineu Prestes 1524, 05508-900, São Paulo, SP, Brazil. jasnam@ibiss.bg.ac.rs.

ABSTRACT

Background: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines.

Methods: DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform.

Results: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands.

Conclusions: These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.

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Related in: MedlinePlus

Characterization of MDA-MB-361 cell line derivatives expressing DCD shRNA. All experiments were repeated using three independent cell clones with essentially identical results. Representative experiments are depicted in the figures. A, Morphology of the cells (upper panel) and immunocytochemical analysis (lower panel) of DCD protein expression. B, Quantitative analysis of colony numbers (≥25 cells/colony) two weeks following plating of the cells. y-axis indicates mean colony counts ± SD; * denotes statistically significant (p < 0.05) differences. Quantitative RT-PCR (C) and immune-blot analysis (D) confirming reduced levels of DCD mRNA and protein, respectively, in DCD shRNA expressing cells. Cellular survival of control pLKO (white bars) and DCD shRNA expressing (black bars) cells following treatment with the indicated concentration of H2O2(E) staurosporine (F) and TNF-α plus cyclohexamide (G). Y axis indicates % surviving cells compared to untreated controls.
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Fig2: Characterization of MDA-MB-361 cell line derivatives expressing DCD shRNA. All experiments were repeated using three independent cell clones with essentially identical results. Representative experiments are depicted in the figures. A, Morphology of the cells (upper panel) and immunocytochemical analysis (lower panel) of DCD protein expression. B, Quantitative analysis of colony numbers (≥25 cells/colony) two weeks following plating of the cells. y-axis indicates mean colony counts ± SD; * denotes statistically significant (p < 0.05) differences. Quantitative RT-PCR (C) and immune-blot analysis (D) confirming reduced levels of DCD mRNA and protein, respectively, in DCD shRNA expressing cells. Cellular survival of control pLKO (white bars) and DCD shRNA expressing (black bars) cells following treatment with the indicated concentration of H2O2(E) staurosporine (F) and TNF-α plus cyclohexamide (G). Y axis indicates % surviving cells compared to untreated controls.

Mentions: To assess the function of DCD in breast cancer cells with high endogenous expression, we generated derivatives of the MDA-MB-361 human breast cancer cell line expressing three different shRNAs against DCD (IBC-I, IBC-II, and IBC-III) using pLKO plasmid-derived lentiviruses. Efficient downregulation of DCD mRNA and protein was confirmed by multiple assays including RT-PCR analyses (2C), immuno-cytochemistry (2A), and immuno-blotting (Figure 2D). Cells expressing DCD shRNAs had significantly reduced colony-forming ability (Figure 2B).Figure 2


Dermcidin exerts its oncogenic effects in breast cancer via modulation of ERBB signaling.

Bancovik J, Moreira DF, Carrasco D, Yao J, Porter D, Moura R, Camargo A, Fontes-Oliveira CC, Malpartida MG, Carambula S, Vannier E, Strauss BE, Wakamatsu A, Alves VA, Logullo AF, Soares FA, Polyak K, Belizário JE - BMC Cancer (2015)

Characterization of MDA-MB-361 cell line derivatives expressing DCD shRNA. All experiments were repeated using three independent cell clones with essentially identical results. Representative experiments are depicted in the figures. A, Morphology of the cells (upper panel) and immunocytochemical analysis (lower panel) of DCD protein expression. B, Quantitative analysis of colony numbers (≥25 cells/colony) two weeks following plating of the cells. y-axis indicates mean colony counts ± SD; * denotes statistically significant (p < 0.05) differences. Quantitative RT-PCR (C) and immune-blot analysis (D) confirming reduced levels of DCD mRNA and protein, respectively, in DCD shRNA expressing cells. Cellular survival of control pLKO (white bars) and DCD shRNA expressing (black bars) cells following treatment with the indicated concentration of H2O2(E) staurosporine (F) and TNF-α plus cyclohexamide (G). Y axis indicates % surviving cells compared to untreated controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4353460&req=5

Fig2: Characterization of MDA-MB-361 cell line derivatives expressing DCD shRNA. All experiments were repeated using three independent cell clones with essentially identical results. Representative experiments are depicted in the figures. A, Morphology of the cells (upper panel) and immunocytochemical analysis (lower panel) of DCD protein expression. B, Quantitative analysis of colony numbers (≥25 cells/colony) two weeks following plating of the cells. y-axis indicates mean colony counts ± SD; * denotes statistically significant (p < 0.05) differences. Quantitative RT-PCR (C) and immune-blot analysis (D) confirming reduced levels of DCD mRNA and protein, respectively, in DCD shRNA expressing cells. Cellular survival of control pLKO (white bars) and DCD shRNA expressing (black bars) cells following treatment with the indicated concentration of H2O2(E) staurosporine (F) and TNF-α plus cyclohexamide (G). Y axis indicates % surviving cells compared to untreated controls.
Mentions: To assess the function of DCD in breast cancer cells with high endogenous expression, we generated derivatives of the MDA-MB-361 human breast cancer cell line expressing three different shRNAs against DCD (IBC-I, IBC-II, and IBC-III) using pLKO plasmid-derived lentiviruses. Efficient downregulation of DCD mRNA and protein was confirmed by multiple assays including RT-PCR analyses (2C), immuno-cytochemistry (2A), and immuno-blotting (Figure 2D). Cells expressing DCD shRNAs had significantly reduced colony-forming ability (Figure 2B).Figure 2

Bottom Line: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines.We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice.Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Institute of Biomedical Sciences - University of São Paulo, Av Lineu Prestes 1524, 05508-900, São Paulo, SP, Brazil. jasnam@ibiss.bg.ac.rs.

ABSTRACT

Background: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines.

Methods: DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform.

Results: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands.

Conclusions: These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.

Show MeSH
Related in: MedlinePlus