Quantitative Mass Spectrometry Identifies Drug Targets in Cancer Stem Cell-Containing Side Population

Abstract

A multifaceted approach is presented as a general strategy to identify new drug targets in a breast cancer stem cellcontaining side population. The approach we have utilized combines side population cell sorting and stable isotope labeling by amino acids in cell culture with mass spectrometry to compare and identify proteins with differential expression profiles between side population cells, know to be enriched in cancer stem cells, and nonside population cells, which are depleted in cancer stem cells, for two breast cancer cell lines, MCF7 and MDA-MB231. Almost 900 proteins were quantified, and several important proteins in cell cycle control and differentiation were found to be upregulated in the cancer stem cell-containing side population. Most interestingly, a splice isoform of pyruvate kinase M2 as well as peroxiredoxin 6 were found to be downregulated.The differential levels of three of these proteins, thymosin ␤4 (TB4), proliferation-associated protein 2G4, and SIAH-interacting protein, were validated using Western blot. Furthermore, functional validation provided clear evidence that elevated TB4 expression contributes to drug resistance in the stem cell population. Small interfering RNA silencing of TB4 led to a loss of chemoresistance in two separate breast cancer populations. These proteins likely contribute to resistance in the cancer stem cellcontaining side population, and their altered expression in a tumor causes clinical resistance to chemotherapy. The ability to perform quantitative mass spectrometry has enabled the identification of a series of proteins that could serve as future therapeutic targets. STEM CELLS 2008;26: 3037-3046 Disclosure of potential conflicts of interest is found at the end of this article.

Ovarian cancer stem-like side-population cells are tumourigenic and chemoresistant

“…Our study indicates that ovarian cancer cells are heterogeneous. SP cells are highly proliferative compared with non-SP cells; SP xenogeneic transplant mice grew more tumours than non-SP xenogeneic transplant mice, as shown from other studies (Chiba et al, 2008;Harris et al, 2008;Steiniger et al, 2008). SP cells have characteristics of cancer stem-like cells.…”

High expression of leptin receptor leads to temozolomide resistance with exhibiting stem/progenitor cell features in gliobalastoma

“…Many studies demonstrated that selection of chemotherapyresistant cells by using anticancer drugs would be an effective means to enrich the CSC-like subpopulation, 28,29 and CSCs indeed possessed intrinsic properties that mediated their resistance to chemotherapy. 30,31 Lzumiya et al found that 5-FU-pretreated pancreatic cancer cells expressed enhanced levels of the stemness genes OCT4 and NANOG, 32 and exhibited a so-called CSC-like phenotype, including a sphere-forming capability.…”

Side Population Cell Level in Human Breast Cancer and Factors Related to Disease-free Survival

“…The breast cancer SP cells in vivo have been shown to be more tumourigenic than NSP cells when tumor cell cultures in immunodeficient mice (Patrawala et al, 2005;Steiniger et al, 2008;Yin et al, 2008). In Zhou et al (2008) study, the sphere cells cultured from SP cells of breast cancer cell MCF7 were enriched in CSC surface markers CD44+CD24-and had higher tumorigenicity.…”

“…It implied the sphere cells enrich breast cancer stem/ progenitor cells and preferentially inhibited by NF-κβ pathway inhibitors compared to their non-stem cell counterparts. Studies have demonstrated that MCF7 SP in the mammary gland are more clinically relevant doses of radiation resistant (Woodward et al, 2007) and clinical resistance to chemotherapy (Steiniger et al, 2008;Yin et al, 2008) than the NSP cell population. These properties would suggest that SP cells may also play a key role in breast cancer progression.…”