Targeting breast cancer stem cells

Abstract

The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of the tumor. Although, the CSC hypothesis does not directly address the cell of origin of cancer, it is postulated that tissue-resident stem or progenitor cells are the most common targets of transformation. Clinically, CSCs are predicted to mediate tumor recurrence after chemo- and radiation-therapy due to the relative inability of these modalities to effectively target CSCs. If this is the case, then CSC must be efficiently targeted to achieve a true cure. Similarities between normal and malignant stem cells, at the levels of cell-surface proteins, molecular pathways, cell cycle quiescence, and microRNA signaling present challenges in developing CSC-specific therapeutics. Approaches to targeting CSCs include the development of agents targeting known stem cell regulatory pathways as well as unbiased high-throughput siRNA or small molecule screening. Based on studies of pathways present in normal stem cells, recent work has identified potential "Achilles heals" of CSC, whereas unbiased screening provides opportunities to identify new pathways utilized by CSC as well as develop potential therapeutic agents. Here, we review both approaches and their potential to effectively target breast CSC.

Figure 1

High‐throughput approaches to target breast CSC. A) Breast CSC can be isolated by sorting using the Aldefluor assay or CD44+CD24− populations (red box) or via culturing as mammospheres. Within these populations, the CSC frequency is enriched with some percentage of non‐tumorigenic bulk cells remaining. B) To screen the CSC population, cells are plated in 384‐well plates in conditions that maintain tumor‐initiation capacity, such as in serum‐free suspension (mammosphere) or in EGF‐ and FGF‐containing media on laminin‐coated plates (Pollard et al., 2009). Small molecule compound collections are added to plates at one compound/well, typically at high nanomolar or low micromolar concentrations. Additionally, the whole genome or selected gene sets (kinases, phosphatases, ‘druggable’) can be targeted using either siRNA or retroviral or lentiviral shRNA libraries with usually 4–5 constructs per gene. shRNA libraries can provide long‐term knockdown whereas siRNA knockdown is more transient. Cellular endpoints can include growth inhibition (Gupta et al., 2009), flow cytometry (Krutzik and Nolan, 2006), sphere formation (Diamandis et al., 2007), or cell migration (Wurdak et al., 2010) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Figure 2

Validation of potential “hits” as anti‐CSC agents. A) Traditionally, “hits” are identified using a Z‐ or B‐score for each compound/RNA and at least 3 standard deviations (green line) from the mean (blue line). Hits (red dots) are “cherry‐picked” for validation studies (right) at additional doses to show specificity for CSC (blue circles) and not normal stem cells (red triangles). B) To confirm validated “hits” target CSCs, activity must be validated using primary tumor xenografts in immunocompromised mice. In the advanced setting (top), cells enriched for breast CSC are injected into mice, allowed to grow to a palpable size and then mice are treated with candidate drugs or siRNA/shRNA. A CSC‐specific agent (blue triangles) would have minimal effect in this assay since tumor growth is driven primarily by progenitors and not CSC. However, an agent that targets CSC and the bulk tumor cells (red squares) would show dramatic tumor reduction. This effect could be achieved using a CSC‐specific agent and a chemotherapeutic agent targeting the bulk population. In an early (i.e. adjuvant) setting (bottom), treatment is initiated soon after injection of CSC‐enriched cells. A selective anti‐CSC agent (blue triangles) would be predicted to have a much greater effect when administered in this setting (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).