The concept of cancer stem cells (CSCs) describes that tumors contain a small proportion of self-renewing and pluri-potent cells that are responsible for initiating and maintaining tumor growth [
1]. This concept is well established in leukemia and has also been reported in a few solid tumor types [
2‐
4]. Recent studies further confirm that a specific cell population is responsible for the initiation and growth of solid tumors [
5‐
7]. These cells usually express high levels of multiple drug resistant gene (MDR1) [
8] and ATP binding cassette (ABC) transporter [
9] and are therefore resistant to chemotherapy and considered as the major source of drug-resistance in tumors. Moreover, it has been demonstrated that CSCs are responsible for metastasis [
7,
10,
11], which is another major cause of cancer-related death. CSCs are thus regarded as an essential target for future advanced cancer therapy.
To achieve the goal of effective treatment of CSCs, identifying specific therapeutic targets is vital. Apart from high throughput screening methods such as microarrays, identifying novel targets of inhibitors or natural drugs is an alternative. A few natural compounds are reported to have inhibitory effects on CSCs [
12‐
14]. These products are valuable for future CSC targeted therapy as they are normally less toxic than chemotherapeutic drugs. For example, vitamin E isotype gamma tocotrienol (γ-T3) was shown to be effective at inhibiting cancer cell growth in several solid tumor models through apoptosis or cell stress related pathways [
15‐
18]. In CSCs, Ling and colleagues reported that γ-T3 could effectively inhibit CSC growth in prostate cancer
in vitro and
in vivo [
19]. They also showed that the CD44 expression of the CSCs was decreased by γ-T3 treatment. CD44 is one of the important epithelial CSC markers, suggesting γ-T3 may affect the stemness of prostate CSCs. However, the detailed mechanism of how γ-T3 suppresses CD44 expression and prostate CSC growth remains unknown. In addition, it is still not clear whether the reduction of CD44 expression was through γ-T3 directly interacting with CD44 or through an indirect interaction with other molecules.
Previously, a study reported that γ-T3 inhibited STAT3 phosphorylation and JAK/STAT pathway activation in different melanoma cell lines, resulting in apoptosis of the cancer cells [
20]. This inhibition was through the induction of SHP1 expression by γ-T3, suggesting that SHP1 was a target of γ-T3 [
20]. However, whether this is the case in CSCs has not been reported or whether there are any new targets in JAK/Stat pathway for γ-T3 remains unknown. In JAK/Stat pathway, there are two very closely related proteins SHP1 and SHP2, they share highly similar structures and sequences. Both of them have two Src homology 2 domains (SH2) that bind to several tyrosine-phosphorylated proteins [
21‐
23]. For biological function, however, SHP1 plays a dominant negative regulation role in the pathway [
24,
25] while SHP2 plays a major positive role [
26‐
28]. Phosphorylation of SHP2 activates associate proteins such as Grb2 and Gab2 and form a protein complex SHP2/Grb2/Gab2. This complex then activates the downstream target RAS and other components of the RAS/ERK pathways [
29‐
31]. SHP2 is encoded by PTPN11, a proto-oncogene in hematologic cells [
32]. Mutation of PTHN11 has been associated with juvenile myelomonocytic leukemias, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, lung cancer, and colorectal cancer [
33]. SHP2 protein levels are elevated in some cancers including cervical cancer [
34] and approx 72 % of breast cancer cell lines [
35]. Many cell types express SHP2 however, SHP1 is expressed in a restricted number of cell types [
36,
37]. These data indicate that compared with SHP1, SHP2 is more likely to be an onco-protein involved in cancer development. Indeed, several studies have shown that inhibition of SHP2 can retard cancer cell growth [
38]. Inhibition of SHP2 gene expression with shRNA was also associated with cell transformation from mesenchymal to epithelial cells, indicating a promoting role of SHP2 in carcinogenesis [
39]. A recent study has shown that SHP2 plays an essential role in the initiation, progression, and metastasis of breast cancer by activating stemness-associated transcription factors such as c-Myc and ZEB1 [
40], further demonstrating its oncogenic role in cancer stem cells. However, there is no report on if γ-T3 targets SHP2 in any cancer types.
Breast cancer is the leading cause of cancer related death among women. Though some studies have shown that using γ-T3 can effectively induce apoptosis or cell cycle arrest in breast cancer cells [
41,
18], there is no report on γ-T3 treating breast CSCs. Particularly, there is no report exploring the potential targets of SHP1 and SHP2 in breast CSCs. In this study, we have demonstrated that γ-T3 had a broad inhibitory effect on human epithelial CSCs including those from breast, colon, and cervical cancers. We found that apart from the effect on SHP1, γ-T3 also targeted SHP2 in breast cancer and that the γ-T3 inhibitory effect on CSC growth was through the RAS/ERK pathway. Moreover, we report here that the level of phosphorylated SHP2 protein increases in breast CSCs, compared with their parental cancer cells, suggesting that SPH2 may play an important role in breast CSC growth and may be considered as a potential therapeutic target for breast CSCs.