Organ-specific adaptive signaling pathway activation in metastatic breast cancer cells

Abstract

Breast cancer metastasizes to bone, visceral organs, and/or brain depending on the subtype, which may involve activation of a host organ-specific signaling network in metastatic cells. To test this possibility, we determined gene expression patterns in MDA-MB-231 cells and its mammary fat pad tumor (TMD-231), lung-metastasis (LMD-231), bone-metastasis (BMD-231), adrenal-metastasis (ADMD-231) and brain-metastasis (231-BR) variants. When gene expression between metastases was compared, 231-BR cells showed the highest gene expression difference followed by ADMD-231, LMD-231, and BMD-231 cells. Neuronal transmembrane proteins SLITRK2, TMEM47, and LYPD1 were specifically overexpressed in 231-BR cells. Pathway-analyses revealed activation of signaling networks that would enable cancer cells to adapt to organs of metastasis such as drug detoxification/oxidative stress response/semaphorin neuronal pathway in 231-BR, Notch/orphan nuclear receptor signals involved in steroidogenesis in ADMD-231, acute phase response in LMD-231, and cytokine/hematopoietic stem cell signaling in BMD-231 cells. Only NF-κB signaling pathway activation was common to all except BMD-231 cells. We confirmed NF-κB activation in 231-BR and in a brain metastatic variant of 4T1 cells (4T1-BR). Dimethylaminoparthenolide inhibited NF-κB activity, LYPD1 expression, and proliferation of 231-BR and 4T1-BR cells. Thus, transcriptome change enabling adaptation to host organs is likely one of the mechanisms associated with organ-specific metastasis and could potentially be targeted therapeutically.

Keywords: DMAPT; NF-kB; TMEM47; brain metastasis; breast cancer.

Figure 1. Validation of genes differentially expressed in brain metastatic cells

A) qRT-PCR analysis of select genes in parental, tumor-derived, and organ-specific metastatic cells. β-actin was used as a normalization control. B) Protein-protein interaction network of two genes expressed preferentially in 231-BR cells. Data were generated using STRING network [31]. Arrow indicates proteins involved in neuronal signaling. C) TMEM47 expression is elevated in another brain-metastasis variant of MDA-MB-231 cells. This variant was derived from BMD-231 cells. D) TMEM47 expression in MCF-7HER2 and its brain metastatic variant.

Figure 2. Prognostic value of genes overexpressed in 231-BR and ADMD-231 cells

A) Elevated expression of 231-BR overexpressed genes (TMEM47, LYPD1, CD96, TFAP2C, EEF1A2, DDX, MYH10, HOXB5, NINJ2, SERPINF1, CPE, MAGEC2, CTLA3, C17orf70, ZNF704, NCKAP1L, and TIE1) in primary breast tumor is associated with poor recurrence-free survival among patients with basal breast cancer. Patients were split by median to classify into high or low expressers. B) Elevated expression of 231-BR specific genes in luminal B breast cancer is also associated with poor recurrence-free survival. C) TMEM47 overexpression is associated with poor brain metastasis-free survival. D) Elevated expression of ADMD-231 overexpressed genes (CYB5R2, TAGLN, HAND1, RAB3IL1, TRMT12, TSPAN8, MMP3, STXBP6, AP1S2, and HSPB8) in primary tumor is associated poor recurrence-free survival among patients with basal breast cancer. E) ADMD-231 overexpressed genes are also associated with poor distant metastasis-free survival among patients with basal breast cancer.

Figure 3. Ingenuity pathway analysis of genes differentially expressed in 231-BR cells

A) Major signaling pathways in 231-BR cells. B) 231-BR cells show activation of SRC-ERK-growth hormone network. C) NF-κB signaling network is active in 231-BR cells. Genes labeled in red are overexpressed, whereas genes in green are expressed at lower levels in 231-BR cells compared with other metastatic cells.

Figure 4. Ingenuity pathway analysis of genes differentially expressed in ADMD-231 cells

A) Notch, FXR/RXR and LXR/RXR networks involved in steroidogenesis similar to adrenal gland are the major pathways in ADMD-231 cells. B) ADMD-231 cells show activation of Notch-ERK-AKT network. C) NF-κB signaling network is active in ADMD-231 cells.

Figure 5. Elevated NF-κB activity in 231-BR cells compared with parental cells

A) DMAPT but not netropsin (netro) inhibited NF-κB DNA binding activity in 231-BR cells. Supershift assays showed p50:p65 NF-κB complex in 231-BR cells. B) DMAPT (10 μM) reduced CXCL1 and LYPD1 but not TMEM47 expression. qRT-PCR was performed to measure mRNA levels. * P values MD-231P versus 231-BR; ** P values untreated 231-BR versus DMAPT-treated 231-BR cells. C) DMAPT inhibited proliferation of 231-BR cells.

Figure 6. 4T1-BR cells displayed elevated NF-κB activity compared with 4T1 cells

A) DMAPT inhibited NF-κB activity in 4T1-BR cells. Note lower AP-1 DNA binding activity in 4T1-BR cells compared with 4T1 cells. B) DMAPT inhibited 4T1-BR cell proliferation.