Increasing CD44+/CD24(-) tumor stem cells, and upregulation of COX-2 and HDAC6, as major functions of HER2 in breast tumorigenesis

Abstract

Background: Cancer cells are believed to arise primarily from stem cells. CD44+/CD24(-) have been identified as markers for human breast cancer stem cells. Although, HER2 is a well known breast cancer oncogene, the mechanisms of action of this gene are not completely understood. Previously, we have derived immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a breast epithelial cell type with stem cell phenotypes after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of HER2/C-erbB2/neu. Recently, we found that M13SV1R2 cells became non-tumorigenic after growing in a growth factor/hormone-deprived medium (R2d cells).

Results: In this study, we developed M13SV1R2N1 under the same growth factor/hormone-deprived condition (R2N1d cells). This provides an opportunity to analyze HER2 effect on gene expression associated with tumorigenesis by comparative study of R2d and R2N1d cells with homogeneous genetic background except HER2 expression. The results reveal distinct characters of R2N1d cells that can be ascribed to HER2: 1) development of fast-growing tumors; 2) high frequency of CD44+/CD24(-) cells (~50% for R2N1d vs. ~10% for R2d); 3) enhanced expression of COX-2, HDAC6 mediated, respectively, by MAPK and PI3K/Akt pathways, and many genes associated with inflammation, metastasis, and angiogenesis. Furthermore, HER2 expression can be down regulated in non-adhering R2N1d cells. These cells showed longer latent period and lower rate of tumor development compared with adhering cells.

Conclusions: HER2 may induce breast cancer by increasing the frequency of tumor stem cells and upregulating the expression of COX-2 and HDAC6 that play pivotal roles in tumor progression.

Figure 1

Derivation of a tumorigenic cell line (R2N1d) and characteristics of R2N1d cells. A, a diagram showing the development of R2d cells which are non-tumorigenic and R2N1d cells which are highly tumorigenic. Both R2d and R2N1d cells were derived from the same immortal cell line M13SV1 and cultured for >10 passages under same cell culture condition before experiment using MSU-1 medium without growth factors and hormones (except 5% FBS). B, expression of ER-α and HER2 in R2N1d cells by immunocytochemical staining (green fluorescence). Cell nuclei were stained with DAPI and recognized as blue fluorescence (top figure, image observed under fluorescence microscope; bottom figure, the mergence of fluorescence and phase images, scale bar = 100 μm). C, R2N1d cells were labelled with antibodies against CK-18, CK-19, Msi1, Notch-1, Notch-4 and Oct-4 for immunophenotyping by flow cytometric analysis. The open histograms indicate background signal and shaded histograms showing positive reactivity.

Figure 2

Functional analysis of genes significantly up-regulated in R2N1d cells compared with R2d cells. A, genes involved in top ten of GeneGo pathway maps and top ten GeneGo process networks by MetaCore analysis. B, number of up-regulated genes belongs to 5 functional categories. Eight genes are at once correlated with metastasis, inflammation and angiogenesis, i.e. TNFRSF12A, CEACAM1, PLAU, HIF1A, IL8, HMOX1, VEGFC and IL1B, whereas two genes are correlated with adhesion, metastasis, and migration, i.e. CD44 and LAMC1.

Figure 3

HER2 effects on COX-2 and HDAC6 expression and cell invasion. A, the effect of AG825 (a HER2 tyrosine kinase inhibitor, 25 μM), U0126 (a highly selective inhibitor of MAPK/ERK kinase, 10 μM) or NS398 (a COX-2 inhibitor, 100 μM) treatment on COX-2 protein expression in R2N1d cells by flow cytometry analysis. B, the effect of AG825, U0126 or NS398 treatment on COX-1 and COX-2 expression in R2N1d cells by western blotting analysis. C, the expression of HDAC6 in R2d and R2N1d cells by flow cytometry analysis. D, the effect of AG825 (25 μM) and LY294002 (10 μM) (a PI3K inhibitor) treatment on HDAC6 expression in R2N1d cells by western blotting analysis. E, the effect of AG825 (25 μM) treatment for 24 hr on HER2 and HDAC6 expression in R2d and R2N1d cells by RT-PCR analysis. F, the effect of HER2 inhibitor, AG825 (25 μM), treatment for 24 hr on invasion ability of R2N1d cells, assayed by using the invasion chamber. R2d cells were also included in the experiment.

Figure 4

Tumorigenicity of R2d and R2N1d cells, and genes expressed by tumors developed by R2N1d cells. A, the development of tumors in immune-deficient mice after inoculation with different numbers of R2d or R2N1d cells (1 × 10⁵, 1 × 10⁶ or 1 × 10⁷ cells per site) for 4 months (R2d) or 4 weeks (R2N1d). The average size of tumors for each treatment are also determined. B, histological sections of resected tumor revealed sheets of polygonal cells with high nucleus/cytoplasm (N/C) ratio. C, these tumor cells expressed Ki67 (90%), VEGF (score 4), COX-2 (score 4) and MMP-9 (score 4). The length of the scale bar of these photos is 100 μm.

Figure 5

Expression of breast cancer stem cell markers CD44⁺/CD24^-/low in R2d and R2N1d cells and Modulation of HER2, Oct-4, AKT and HDAC6 expression in R2N1 cells by cell culture condition. A, higher frequency of CD44⁺/CD24^-/low cells was found in R2N1d cells than R2d cells (~50% vs. ~10%). R1 region denotes a small population of CD44^high cells which was increased in R2N1d cells. The fluorescence cut off level: high expression was fluorescence intensity > 10³; negative or low expression was fluorescence intensity < 10². B, CD44⁺/CD24^- cells sorted by flow cytometry tend to show contact-insensitive growth in confluent culture and gave rise to non-adherent cells in suspension. By flow cytometric analysis (C, E), and by western blotting (D), adherent cells and re-attached cells were found to be HER2⁺/OCT4⁺/AKT⁺/HDAC6⁺, whereas non-adherent cells were HER2^-/OCT4⁺/AKT ^-/HDAC6^-. The length of the scale bar of these photos is 100 μm.

Figure 6

Tumorigenicity of 3 types of R2N1d cells in immune-deficient mice. Adherent (CD44⁺/CD24^- sorted), non-adherent and re-attached R2N1d cells were inoculated subcutaneously into nude mice for tumor development. Only those mice inoculated with adherent and reattached R2N1d cells showed visible and palpable tumors 4 weeks after inoculation; Mice inoculated with non-adherent R2N1d cells developed tumors after 24 weeks. Histological sections of resected tumor harvested at 4 weeks (from adherent and reattached cells) or 24 weeks (from non-adherent cells) showed HER2 expression. The length of the scale bar of these photos is 100 μm.

Figure 7

A diagram depicting the change in HER2 expression and tumorigenicity of R2N1d (CD44⁺/CD24^- sorted) cells due to cell culture condition. Adherent R2N1d cells were OCT4⁺/HER2⁺ and highly tumorigenic (tumor developed in 1 month after inoculation), the non-adherent R2N1d cells detached from confluent R2N1d cells in suspension were OCT4⁺/HER2^- and took longer time (6 months) to develop tumor at lower frequency. The reattached R2N1d cells from non-adherent cells were similar to parental adherent cells in OCT4⁺/HER2⁺ expression and tumor development.