Tight correlation between expression of the Forkhead transcription factor FOXM1 and HER2 in human breast cancer

Abstract

Background: FOXM1 regulates expression of cell cycle related genes that are essential for progression into DNA replication and mitosis. Consistent with its role in proliferation, elevated expression of FOXM1 has been reported in a variety of human tumour entities. FOXM1 is a gene of interest because recently chemical inhibitors of FOXM1 were described to limit proliferation and induce apoptosis in cancer cells in vitro, indicating that FOXM1 inhibitors could represent useful anticancer therapeutics.

Methods: Using immunohistochemistry (IHC) we systematically analysed FOXM1 expression in human invasive breast carcinomas (n = 204) and normal breast tissues (n = 46) on a tissue microarray. Additionally, using semiquantitative realtime PCR, a collection of paraffin embedded normal (n = 12) and cancerous (n = 25) breast tissue specimens as well as benign (n = 3) and malignant mammary cell lines (n = 8) were investigated for FOXM1 expression. SPSS version 14.0 was used for statistical analysis.

Results: FOXM1 was found to be overexpressed in breast cancer in comparison to normal breast tissue both on the RNA and protein level (e.g. 8.7 fold as measured by realtime PCR). We found a significant correlation between FOXM1 expression and the HER2 status determined by HER2 immunohistochemistry (P < 0.05). Univariate survival analysis showed a tendency between FOXM1 protein expression and unfavourable prognosis (P = 0.110).

Conclusion: FOXM1 may represent a novel breast tumour marker with prognostic significance that could be included into multi-marker panels for breast cancer. Interestingly, we found a positive correlation between FOXM1 expression and HER2 status, pointing to a potential role of FOXM1 as a new drug target in HER2 resistant breast tumour, as FOXM1 inhibitors for cancer treatment were described recently. Further studies are underway to analyse the potential interaction between FOXM1 and HER2, especially whether FOXM1 directly activates the HER2 promoter.

Figure 1

Elevated expression of FOXM1 mRNA in breast cancer cell lines. Semiquantitative realtime PCR (LightCycler) of FOXM1 expression was performed on reverse transcribed RNA from non-malignant (white bars) and malignant cell lines (black bars). A significant difference in expression between both groups was detected (P = 0.048), whereas classification of malignant breast cell lines into oestrogen receptor (ER) positive and negative revealed no significant coherence with FOXM1 expression (P = 0.655; two-tailed Mann-Whitney U-test).

Figure 2

Upregulation of FOXM1 expression in primary breast cancer. A collection of paraffin-embedded breast carcinomas (T; n = 25) and normal breast tissues (N; n = 12) was analysed for FOXM1 expression by semiquantitative realtime PCR. In total, we detected a strong upregulation at transcript level in the tumourous tissues as compared with normal breast tissues.

Figure 3

Immunohistochemical expression of FOXM1 in normal breast tissue as well as in non-invasive and invasive breast tumours using a tissue microarray. (A, B) Normal breast tissue is detected with very weak FOXM1 expression in the nucleus (IRS = 1) and weak FOXM1 expression in the cytoplasm (IRS = 4). (C, D) In ductal carcinoma in situ of high grade type nuclear FOXM1 expression is a little more intense (IRS = 2) than in normal breast tissue whereas the cytoplasmic FOXM1 expression is strong (IRS = 9). (E, F) In invasive breast carcinoma FOXM1 expression was often stronger in the nucleus (IRS = 3) as well as in the cytoplasm (IRS = 12) compared with normal breast tissue and ductal carcinoma in situ. (G, H) In tubular breast carcinoma, a rare variant of invasive breast carcinoma with a more favourable prognosis than invasive ductal breast carcinoma, nuclear and cytoplasmic FOXM1 expression (each IRS = 3) was weaker than in most invasive ductal breast carcinomas and stronger than in most normal breast tissues. Magnifications: A, C, E, G: 100×; B, D, F, H: 400×.

Figure 4

Breast cancer patients expressing nuclear FOXM1 show a tendency towards unfavourable prognosis. Univariate Kaplan-Meier analysis was performed on basis of expression results from a tissue microarray. Patients with weak or absent nuclear FOXM1 expression (IRS = 0–1) displayed improved overall survival estimation (upper graph) as compared to patients with strong nuclear FOXM1 expression (lower graph). Level of significance was only marginal (P = 0.110; univariate log-rank analysis). IRS=Immunoreactive score according to Remmele et al. [23]. Crosses indicate censored patients.