Many different types of tumors contain a subset of stem-like tumor-initiating cells, which drive tumor initiation and recurrence. Thus, therapeutic strategies that selectively target TICs are proposed to lead to the development of successful anti-cancer therapies. The molecular mechanisms underlying tumor initiation and stem-like function of TICs, however, are poorly understood. To understand these mechanisms, we investigated to identify essential regulators for the maintenance of breast TICs. We first identified and characterized TICs and non-TICs isolated from a mouse breast cancer model. We demonstrated that the breast TICs, which are highly enriched in the CD29highCD24+ subpopulation, drive tumor initiation and possess stem-like capabilities including self-renewal, heterogeneous differentiation, and bipotency, compared with other subpopulations. Comparison of gene expression between breast TICs and non-TICs revealed that FGFR2 is upregulated in the breast TICs. Our studies suggested that FGFR2 is essential in sustaining the breast TIC pool through promotion of self-renewal and maintenance of bipotency of TICs. Our studies contribute to the characterization of stem-like function of breast tumor-initiating cells and to the understanding of the associated molecular mechanisms. In addition, our results targeting FGFR2 suggest that there are specific targets that may be used to successfully eliminate TICs in human cancers.
This is the first report demonstrating convergence of two recent findings: FGFR2 function in breast cancer and in normal mammary stem cell maintenance. FGFR2
was recently identified as a risk factor in breast cancer from several genome-wide association studies for breast cancer 
. Indeed, FGFR2
is amplified and overexpressed in 4–12% of human breast cancers 
. A mouse mammary tumor virus (MMTV) insertional mutagenesis screen for genes associated with mammary cancer also identified FGFR2
. Independent of these roles of FGFR2 in breast cancer, recent studies in mammary gland development revealed that FGFR2 is essential in the maintenance of terminal end buds (TEBs) where mammary stem cells are active during mammary gland development 
. Although independent studies demonstrated that FGFR2 plays a significant role in normal or malignant breast tissue, none of these findings made FGFR2 signaling as a functional link between the normal mammary stem cells and malignant mammary stem cells.
It has been speculated that the self-renewal regulators in normal stem cells may be shared by malignant stem cells or TICs, since malignant stem cells have been proposed to derive from the transformation of normal stem or progenitor cells through dysregulated self-renewal 
. The potential role of FGFR2 in mammary stem cell maintenance in TEBs 
is further supported by the requirement of FGF10–FGFR2IIIb signaling for embryonic and postnatal mammary gland development 
. In addition, a role for FGFR2 in stem cell maintenance is reinforced by the evidence of FGFR2 functions in a variety of stem cells. These functions include self-renewal and proliferation of the undifferentiated state of multipotent trophoblast stem cells during embryogenesis 
, proliferation of osteogenic stem cells 
, and maintenance of self-renewal and undifferentiated growth of human embryonic stem cells 
Activating somatic mutations of FGFR2
have been reported in some cancers including lung squamous cell carcinoma, gastric cancer, cervical carcinoma, and endometrial carcinoma, implicating its role in cancer development 
. We sequenced the regions of exons (exon 3, 6, 7, 8, 9, 12, and 14) in which somatic mutations of FGFR2
have been reported in the other carcinoma cases 
. Although we found one SNP, rs1047100 (696A→G) in exon 6 of FGFR2
in 60% of the 30 primary human breast tumors analyzed, we failed to detect any of the previously described FGFR2
somatic mutations in either 30 primary human breast tumors or in MMTV-PyMT tumors (unpublished data), suggesting that FGFR2
somatic mutations may be an infrequent event in breast cancer. However, several recent genome-wide association studies demonstrated that germ-line polymorphisms in intron 2 of FGFR2
are associated with breast cancer susceptibility 
emphasizing the importance of FGFR2
in breast cancer. The mechanism by which FGFR2
functions as a risk factor in breast cancer, however, remains unknown. The present findings that FGFR2 is an essential regulator for the maintenance of breast TICs may answer questions, at least in part, in breast cancer.
Alternative splicing in the third immunoglobulin-like domain of FGFR2 mRNA results in the formation of FGFR2IIIb and FGFR2IIIc isoforms (Figure S4A
). This differential splicing determines the ligand specificity of FGFR2 in a tissue specific manner 
. Epithelial cells express FGFRIIIb, whereas mesenchymal cells express FGFR2IIIc (Figure S4A
). To investigate which isoform of FGFR2 is present in MMTV-PyMT tumors, we sequenced the cDNAs generated from mRNA regions common to both FGFR2IIIb and FGFR2IIIc isoforms (Figure S4A and S4B
). This analysis revealed that MMTV-PyMT tumors express FGFR2IIIb isoform which is exclusively expressed by epithelial cells. Further studies will be required to understand a functional link between the FGF ligands (to the particular FGFR2 isoform) and the FGFR2 roles in TIC maintenance.
TKI258 is an orally bioavailable kinase inhibitor of the FGFRs. Its activity against VEGFR and PDGFR, along with the FGFR inhibitory activity, is responsible for the potent anti-angiogenic component of the compound. TKI258 is now being investigated in phase III and II clinical trials in renal cell carcinoma, breast cancer, myeloma and urothelial cancer. In our studies we have shown that use of TKI298 leads to an inhibition of tumor growth in this mouse model of breast cancer and this data could serve as a predictor for its use in breast cancer patients. Given the multitargeted nature of TKI258, our results emphasize the need to develop FGFR2 specific inhibitors.
Our gene expression comparison between breast TICs and differentiated non-TICs led to the identification of upregulated genes in breast TICs, including FGFR2
, and CDC42BPG
. These genes have been shown to be involved in mammary carcinogenesis, development and stem cell activities 
. However, the expression of these genes has not been previously investigated regarding their involvement in maintenance of tumor-initiating cells. Further studies will also be required to understand any functional link between FGFR2 and other genes that were upregulated in TICs. It is possible that the inhibition of other TIC-specific genes along with FGFR2 inhibition produces synergistic anti-tumor activity, more effective at eradicating tumor initiating cells.