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New Cancer Stem Cell Sorting Strategy to Boost Breast Cancer Research

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Review of “Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer” from Stem Cells by Stuart P. Atkinson

Triple-negative breast cancer (TNBC) represents the most aggressive form of breast cancer [1], and suffers from poor prognosis, tumor recurrence and a lack of therapeutic options [2]. These characteristics suggest that cancer stem cells (CSC) may lie at the root of TNBC maintenance and progression, although the lack of an effective means to enrich CSCs from non-CSCs has hampered the detailed analysis of CSCs in this setting [3]. To solve this problem, researchers from the laboratory of Ofer Reizes (Lerner Research Institute, Cleveland, USA) have developed a novel CSC reporter system using a GFP reporter driven by the promoter of NANOG [4], a master regulator of stem cell self-renewal. In this new study published in Stem Cells they highlight the use of this system in the assessment of CSC markers and the mechanisms controlling TNBC initiation and progression [5].

The group first transfected the NANOG-GFP reporter into TNBC cell lines (MDA-MB-231 and HCC70) and following cell sorting (See figure) they found that GFP-positive cells highly resembled CSCs. In vitro culture of GFP-positive cells generated both GFP-positive and GFP-negative cells, demonstrating the development of cellular heterogeneity over time, while they also observed a cell surface marker expression pattern linked to CSCs (CD24−/CD44+/CD49fHi). Additionally, GFP-positive cells displayed an increased mesenchymal phenotype, an increased invasive capacity, and exhibited increased self-renewal, all of which are characteristic of CSCs. GFP-positive cells also had a greater ability to initiate tumorigenesis after transplant into NOD-SCID mice, and resulted in significantly increased tumor growth, and GFP-positive cells isolated from these tumors displayed greater sphere-forming cell frequencies and increased self-renewal capacity when compared to GFP-negative cells.

Finally, the group implemented high-throughput flow cytometric screening to generate a highly pure CSC population and to identify CSC-specific molecular pathways. Alongside the CSC surface markers (CD24−/CD44+/CD49fHi), the researchers found increased JAM-A expression in the GFP-positive fraction compared to GFP-negative cells. JAM-A (or F11R) regulates tight junction assembly and correlates with poor prognosis in patients with invasive breast cancer [6]. Short hairpin RNA mediated inhibition of JAM-A caused a reduction in CSC self-renewal, while upregulation in GFP-negative cells significantly increased the frequency of self-renewing cells, suggesting that JAM-A may represent a target for anti-CSC therapeutics.

This study suggests that application of the NANOG-GFP reporter could represent an important resource towards research into triple negative breast cancer, and assessment in other tissues may also prove this tool to be of wider utility. The high purity sorting facilitated by this reporter should cut through previous problems of heterogeneity and identify bona fide therapeutic targets and prognostic gene signatures.

Discussion Points

  • Will this strategy reveal potentially new druggable targets for TNBC therapy?
  • Will JAM-A be useful as prognostic marker in TNBC patients?
  • Will findings fully reflect the in vivo situation?
  • Will this reporter construct be useful for the isolation of other cancer stem cell populations from other tumor types?
  • Could this reveal a common CSC mechanism which can be targeted?

References

  1. Dent R, Trudeau M, Pritchard KI, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clinical cancer research : an official journal of the American Association for Cancer Research 2007;13:4429-4434.
  2. Metzger-Filho O, Tutt A, de Azambuja E, et al. Dissecting the heterogeneity of triple-negative breast cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2012;30:1879-1887.
  3. Schmitt F, Ricardo S, Vieira AF, et al. Cancer stem cell markers in breast neoplasias: their relevance and distribution in distinct molecular subtypes. Virchows Arch 2012;460:545-553.
  4. Zheng Q, Banaszak L, Fracci S, et al. Leptin receptor maintains cancer stem-like properties in triple negative breast cancer cells. Endocr Relat Cancer 2013;20:797-808.
  5. Thiagarajan PS, Hitomi M, Hale JS, et al. Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer. Stem Cells 2015;33:2114-2125.
  6. McSherry EA, McGee SF, Jirstrom K, et al. JAM-A expression positively correlates with poor prognosis in breast cancer patients. International journal of cancer. Journal international du cancer 2009;125:1343-1351.