Triple negative breast cancer (TNBC) remains an aggressive disease due to the lack of targeted therapies and low rate of response to chemotherapy that is currently the main treatment modality for TNBC. Breast cancer stem cells (BCSCs) are a small subpopulation of breast tumors and recognized as drivers of tumorigenesis. TNBC tumors are characterized as being enriched for BCSCs. Growing evidence has shown that breast cancer stem cells are associated with tumor initiation and metastasis and may play a critical role in chemoresistance. Multiple targets against breast cancer stem cells are now under investigation. Recent advances in the role of breast cancer stem cells in triple negative breast cancer and the identification of cancer stem cell biomarkers have paved the way for the development of new targeted therapies.
1. Cancer Stem Cells in Breast Cancer
BCSCs are a small cell subpopulation among all tumor cells, with unique stem cell characteristics such as self-renewal, high proliferation and differentiation potential to multiple lineages within the breast. BCSCs interact with their tumor microenvironment (TME) as well as on the inducing factors and elements. Markers that have been identified in BCSCs include ALDH1+, CD24− and CD44+. Cells which simultaneously express these markers have the highest tumor initiating and proliferative capacity. Several theories have been proposed about the origin of BCSCs. One of them suggests mutations in dormant normal stem cells results in their transformation to cancer stem cells, a process similar to that of non-stem cells 
Several markers have been used for the isolation and identification of BCSCs. In 2003, Al-Hajj M et al. first described and isolated the BCSC phenotypes CD24− and CD44+ 
. CD24 is a cell surface glycoprotein, which has been shown to play a role in tumor progression and metastasis. As a ligand for P-selectin, CD24 is proposed to interact with endothelial cells and platelets during metastasis and ultimately has been linked to poor prognosis and decreased survival 
. As another cell surface glycoprotein and specific receptor to hyaluronan, CD44 plays a key role in breast cancer adhesion, motion, migration and invasion, all of which have a significant impact on early tumor metastasis. 
ALDH1 is a recently described BCSC marker which belongs to a family of cytosolic enzymes involved in oxidation of intracellular aldehydes that convert retinaldehydes to retinoic acid 
Six molecular subtypes of TNBC have been proposed: immunomodulatory, mesenchymal, mesenchymal stem-like, luminal androgen receptor and two basal-like subtypes 
. The CD44+/CD24− lineage cells generally possess a mesenchymal or myoepithelial-like phenotype and are found more peripherally at the tumor edge. ALDH1-expressing BCSCs have a more epithelial or luminal phenotype and are located more centrally in tumors. These characteristics enable effective epithelial-mesenchymal transition and vice versa 
. This explains the increased rate of metastasis in BCSC enriched tumors.
Cancer cells with the CD44+/CD24−/ALDH1+ phenotype in breast cancer can therefore be distinguished from other cancer cells by their stem-like features, ability to maintain survival and the role in cancer invasion and metastasis, particularly in TNBC.
2. Breast Cancer Stem Cell Regulation Pathways in Triple Negative Breast Cancer
TNBC tumors are known as being enriched for BCSCs. Transcription factors, signaling pathways and tumor suppressor genes have played a pivotal role to maintain the state of stemness of BCSCs. The Wnt, Notch, and Shh pathways have been identified as playing an important role in BCSC and TNBC biology 
. Like in many other epithelia, Wnt pathway activity is critical for stem cell self-renewal and multipotency in the breast 
. Although canonical Wnt signaling is the most well-known path to β-catenin stabilization, numerous other signaling pathways also regulate β-catenin stability and transcriptional activity 
. For instance, the β-catenin mediated Wnt pathway has several downstream targets including MMP7 and PTEN, as characterized by Dey and colleagues 
. In TNBC, differential activation of the Wnt pathway correlates with increased MMP7 and decreased expression of PTEN. Patients with increases in MMP7 have a lower rate of pathologic complete response and a high residual disease burden 
. Inhibition of the Wnt pathway leads to decreased proliferation and induction of apoptosis, bringing to light possible therapeutic targets 
. Additionally, wnt10B/β-catenin modulates HMGA2, and expression of HMGA2 has been shown to predict the metastasis rate and relapse-free survival 
. Sulaiman and colleagues found that increased Wnt and histone deacetylase (HDAC) activities are associated with loss of ER and PR expression, poor survival, and increased relapse in patients with invasive breast cancer. Furthermore, in a subset of TNBC cell lines, Wnt signaling and the repression of ER and PR was found to be inversely correlated 
The canonical Notch signaling pathway is initiated by activating Notch receptors upon binding to Serrate- and Delta-like ligands present on the cell membranes of adjacent cells. Notch receptors have intracellular domains that are releases into the nucleus after proteolytic cleavages. These Notch intracellular domains can recruit MAML1 and histone acetyltransferase p300 to form active transcriptional complexes, which is the final regulator of the Notch target genes 
. It was found that the Notch 4, one of the four Notch receptors, is involved in the constitutive ligand-independent activation of Notch 4, and could facilitate the development of mammary adenocarcinoma 
. BCSCs ectopically expressing Notch 4 in TNBC have shown increased proliferation and invasiveness, whereas inhibition/knockdown of Notch4 decreases cell proliferation, invasion, tumor volume, and tumorigenicity 
The canonical Shh pathway is activated by releasing its ligands to bind and inhibit transmembrane receptor Patched-1 (PTCH1). PTCH1 can subsequent initiate an intracellular signal cascade to activate of the downstream transcription factors of glioma-associated oncogene (GLI) 
. Common Shh target genes include Cyclin D1/2 
, MYC 
, BCL2 
, VEGF 
, MMP9 
and SOX2 
. The non-canonical Shh pathway, on the other hand, does not reply on the PTCH1 to activate GLI. Instead, it can cross talk with a variety of other signaling cascades 
. Of note, Shh pathway activation is involved in the TME of the CD44+/CD24−/ALDH1+-expressing BCSCs. Shh pathway activation has been found to mediate the self-renewal of BCSCs after radiation or chemotherapy, which, not surprisingly, results in therapy resistance.
The self-renewal mechanism of CSCs, involving the signal transduction pathways noted above and including Wnt, Notch, and Shh 
, is illustrated in Figure 1
A. Further investigation of these signaling pathway functions and their interactions with other pathways can aid the development of CSC-targeted therapy for the treatment of TNBC.
Figure 1. Summary of CSC specific mechanisms and pathways targeting breast cancer stem cells (A) and non-CSC specific mechanisms and pathways targeting breast cancer cells (B).
3. Tumor Metastasis in Triple Negative Breast Cancer
Using CSCs as a prognostic marker in patients with breast cancer has profound clinical significance and has been discussed in one of our prior reviews 
. The prognostic value of these CSC markers in breast cancer, especially the association with metastasis occurrence and survivals, has been summarized in Table 1
. Based on the accumulating evidence, it appears as if combining the three stem cell markers CD44, CD24, and ALDH1 has tremendous value in predicting prognosis, including risk of metastasis and overall survival.
Table 1. Summary of studies on metastasis occurrence and survivals in stem like breast cancer.
|(Abraham et al., 2005) 
||12 (80%) cases with CD44+/CD24−/low phenotype had distant metastasis (p = 0.04). All 5 cases with more than 20% CD44+/CD24−/low tumor cells had osseous metastasis (p = 0.02).
||The percentage of CD44+/CD24−/low tumor cells had no influence on DFS or OS
|(Liu et al., 2007) 
||The IGS * in CD44+/CD24−/low cancer cells was significantly associated with the risk of metastasis regardless of tumor size or lymph-node status (p < 0.05).
||Of patients treated with chemotherapy,
IGS in CD44+/CD24−/low cancer cells was
associated with lower 10-year metastasis-free survival (p < 0.001).
|(Lin et al., 2012) 
||The proportion of CD44+/CD24− tumor cells was correlated with lymph node involvement (p = 0.026).
||The proportion of CD44+/CD24− tumor
cells was significantly associated with DFS (p = 0.002) and OS (p = 0.001).
|(Adamczyk et al., 2014) 
||In patients treated with anthracyclines and taxanes, significantly longer survival was associated with CD44+ phenotype (DFS p = 0.019, OS p = 0.062) and CD44+/CD24− phenotype (DFS p=0.006, OS p = 0.019).
|(Chen et al., 2015) 
||The proportion of CD44+/CD24− tumor cells was significantly associated with lymph node involvement (p = 0.016), distant metastasis (p = 0.001), and recurrence (p = 0.013)
||High CD44+/CD24− phenotype had worse response to chemotherapy (p = 0.001), and worse DFS (p = 0.0012) and OS (p = 0.017)
|(Collina et al., 2015) 
||Only CD44, not CD24, CD133, ALDH1 and ABCG2, was significantly associated with metastases (p = 0.011).
||Among CD44, CD24, CD133, ALDH1 and ABCG2, only CD44 was significantly associated with DFS (p = 0.051).
|(Wang et al., 2017) 
||CD44+/CD24− subtype possessed slightly increased risk of metastasis or recurrence compared with CD44−/CD24− subtype.
||CD44+/CD24− tumor cells were associated with worse OS (p = 0.005).
|(Ma et al., 2017) 
||ALDH1 expression was significantly correlated with tumor stage (p = 0.04).
||ALDH1 expression was associated with shorter RFS (p = 0.01) and OS (p = 0.001).
|(Lee & Kim, 2018) 
||Both cutaneous metastatic cases had high expression of CD44+/CD24− and ALDH1+.
|(Rabinovich et al., 2018) 
||CD44+/CD24− phenotype was associated with a greater risk of relapse (p = 0.011) and a worse outcome (p = 0.019). TNBC was associated with ALDH+ (p = 0.039).
|(Althobiti et al., 2020) 
||The high expression of ALDH1 was significantly associated with poor survival (p < 0.001), and particularly in the luminal B (p = 0.042) and TNBC (p = 0.003) subtypes.
The epithelial–mesenchymal transition (EMT) has been recently recognized to have the ability to convert epithelial cells to mesenchymal cells and subsequently acquire motile and migratory propensity 
. This propensity, as expected, plays a key role for CSCs to metastasize to distant tissues or organs regardless of chemotherapy 
. Several signaling pathways in the TME, including, but not limited to, Wnt, Notch, and Shh, as mentioned earlier, produce transcription factors to regulate tumor growth, invasion, and metastasis. If these tumor cells are present in patient vessels and/or lymphatics, they are called circulating tumor cells (CTCs) and have been hypothesized to be involved in the metastatic process 
. Increasing evidence has demonstrated that CSC markers are conserved without alteration during tumor metastasis, with CTCs serving as intermediate cells from primary tumor cells and the distant metastatic tumor cells 
. BCSCs have been identified in a CTC population among patient peripheral blood samples 
. Accordingly, this finding makes it possible to detect CTCs by using stem cell markers mentioned above 
. These markers, while indicating the stemness of the CTCs, may be used for the early diagnosis of tumor metastasis, prognosis prediction and therapeutic effect monitoring.
It has been shown that TNBC exhibits more traits possessed by CSC than other breast cancer subtypes and is more likely to develop metastases 
. Driven by the aggregation of CD44+ CSC, more CTC clusters and polyclonal metastasis of TNBC were found to be associated with an unfavorable prognosis 
. The epidermal growth factor receptor (EGFR) family has been found to be involved in the regulation of cancer metastasis, including TNBC 
. Interactions between the receptor tyrosine kinases EGFR and metastasis with extracellular matrix (ECM)-binding integrins enhance metastatic colonization in model systems 
. The current evidence supports dynamic crosstalk between CSCs and metastatic site TME, with contributions by surrounding non-stem cells, including stromal cells, immune cells, and ECM, which are crucial for tumor growth after CTCs seed in the metastatic site 
. CSCs in TNBC have been shown to have characteristic their high invasiveness and metastatic behavior, with increased expression of pro-invasive genes, including those for interleukin (IL)-1, IL-6, IL-8, and urokinase plasminogen activator 
All the above mechanisms have been shown to be possibly involved in the high propensity for invasiveness and metastasis of BCSCs in TNBC. Targeting these mechanisms can potentially prevent metastasis and therefore improve survival in TNBC.
4. Chemoresistance in Triple Negative Breast Cancer
TNBC, the most aggressive and deadliest type of breast cancer, lacks a targeted therapy and has therapy-resistance to the majority of chemotherapy regimens. Since none of the current chemotherapies specifically targets CSCs, CSCs serve as a tumor reservoir for self-renewal and proliferation. This causes inevitable tumor invasion and metastasis, which is more common in TNBC than non-TNBC, ultimately resulting in poor prognosis. Studies have shown CSCs are associated with chemoresistance, particularly in TNBC due to the higher propensity of developing stemness compared to those found in non-TNBC 
Doxorubicin (Dox), for instance, is widely used in the treatment of TNBC. However, resistance by tumor cells limits its effectiveness. CSCs have been found to be associated with Dox resistance. Signal transducer and activator of transcription 3 (Stat3) and its downstream pathway can convert non-CSCs to CSCs 
. More recently, a novel signaling pathway that involves Stat3, Oct-4 and c-Myc has been demonstrated to regulate stemness-mediated Dox resistance in TNBC 
. To overcome the mechanism of Dox resistance, WP1066, a Stat3 inhibitor that was more widely studied to treat CNS tumors, is being explored to reduce proliferation of BCSCs in Dox-resistant TNBC 
Since the CD44+/CD24−/ALDH1+ phenotypes have been recognized as BCSC markers in many studies, a hypothetical approach to targeting CSCs against cell surface membrane antigens such as CD44 would be justified 
. Target options include monoclonal, competitive protein/peptide, HD-CD44 crosstalk, and others 
. In addition, ALDH1 can be a possible therapeutic target 
, although only limited in vitro data is available through ALDH1 knockdown 
Immune evasion has also been suggested to result in chemoresistance in TNBC, as well as tumorigenesis in other tumor types. Tumor-infiltrating lymphocytes (TILs) have been shown to be present in some TNBC and non-TNBC breast cancers and have been associated with favorable prognosis 
. Programmed cell death-1 (PD-1) receptor expression on tumor cells and programmed death-ligand 1 (PD-L1) expression on TILs are therefore indicators of the enrichment of the adaptive immune response against tumor cells. Lack of TIL or PD-1/PD-L1 expression in the tumor microenvironment has been suggested to be associated with less favorable prognosis, especially in early-stage breast cancer 
. The blockade of PD-1/PD-L1 with checkpoint inhibitors has become a promising immunotherapy to enhance anti-tumor immunity in TNBC after success in treating other types of cancer and is being widely investigated in many ongoing studies.
The involvement of any of the above mechanisms may result in chemoresistance in TNBC. It is essential to take these mechanisms into account when developing novel targeted therapies to overcome chemoresistance.