Anti-Tumour TreatmentCancer stem cell in breast cancer therapeutic resistance
Introduction
Breast cancer (BC) is one of the most common cancers responsible for approximately 30% of new female cancer cases and ranked as the 2nd cause of cancer-related deaths in annual statistics [1]. The treatment options for BC, including breast-conserving surgery or mastectomy, radiotherapy (RT), chemotherapy (CT), hormone therapy (HT), and other novel therapies, are decided based on the individual features of clinico-pathology. For instance, mastectomy and adjuvant RT are utilized for many early BCs with curative intent. Conventional anticancer drugs can be employed as a single agent or in combinations to minimize the recurrence risk. For women with estrogen receptor positive (ER+) or human epidermal growth receptor positive (HER2+) tumors, tamoxifen or trastuzumab respectively contribute to the substantial improvements in long-term survival rate. These therapeutic options are considered as a milestone in dealing with BC.
However, many BC patients still experienced relapse in a few years and the long-term mortality remains high. The 15-year BC mortality fluctuated between 41.3% and 49.5% regardless of post-mastectomy radiation [2], indicating current therapies blend BC treatment with high degrees of uncertainty in spite of widely applied neoadjuvant therapies. BC is normally treated based on its intrinsic subtypes, which can only partially explain the biology and response to treatment. The failure of treatment to deal with intractable cancer cells has raised a question of whether there is a special population of cells in tumor heterogeneity which exhibit resistant phenotypes that favor the micrometastasis and have the potential to cause recurrence.
For the past few years, cancer stem cell (CSC) model was proposed and has received increasing interest. Collective work has revealed that tumor regeneration could be initiated by these CSCs. They are capable of self-renewal, recapitulating the heterogeneity of original tumors, and differentiating into the whole bulk of a new tumor in immunocompromised mice. Fractional irradiation caused lower level of reactive oxygen species (ROS) in breast cancer stem cells (BCSCs) compared to highly differentiated tumor cells, suggestive of a radioresistant phenotype [3]. Treating BCSCs with a multidrug CT not only increased the expressions of markers in pre-existing BCSCs but also promoted CSC-dependent non-stem cancer cells-to-CSC conversion [4]. As a result, targeting BCSCs seems to be an efficient adjuvant way to improve disease prognosis.
In this review, we summarize the latest studies about cell surface markers and signaling pathways that sustain the stemness of BCSC and discuss the associations of mechanisms behind these traits with BCSC generation, regulation, and transition. More importantly, their implications for future study are evaluated and potential BCSC-targeted strategies are proposed to break through the restriction of current therapies. We believe that the further exploration in this field of research will help researchers effectively identify and target BCSCs in tumors and eventually help doctors and patients achieve an improved response to BC therapy.
Section snippets
Is CSC the culprit of BC therapeutic failure?
The existence of CSCs was first evidenced by Bonnet and Dick [5] in human acute myeloid leukemia. These cells were similar to normal hematopoietic stem cells and can hierarchically differentiate into leukemic clone. The hierarchy resembles the differentiation process of hematopoietic progenitor cells and puts forward the necessity of targeting CSCs in cancer treatment. Based on research findings, a consensus definition of CSC was proposed by American Association for Cancer Research in 2006, and
BCSC surface markers: identification, function, and targeting
BCSC surface markers are initially used to identify and isolate CSCs from BC via flow cytometry. However, outcomes from the latest studies suggested that their activities may also determine the internal diversity among BCSCs. More importantly, as most of BCSC surface markers are membrane receptors, they are critical for the design of novel targeted drugs that would be recognized by BCSCs of interest in order to achieve a more positive response to anticancer treatment. Therefore, in this part,
CD44
CD44 was the first effective surface marker used to identify CSCs. As a cell receptor, CD44 mediates the communication with microenvironment through interacting with extracellular ligands, such as hyaluronan (HA). The interaction of CD44 and HA can stimulate RhoA-specific guanine nucleotide exchange factor-mediated RhoA/Grb2-associated binder-1 signaling or c-Src kinase/Twist/miR-10b/RhoA signaling that are responsible for the activation of the PI3K/AKT signaling pathway [54]. Interestingly,
Integrins (CD29, CD49f, and CD61)
Similar to CD44, integrins are also major cell surface receptors for extracellular ligands, such as fibronectin and laminin. They can heterodimerize with each other and mediate cell adhesion to the extracellular matrix, undertaking bidirectional communications with microenvironment. CD29, CD49f, and CD61 which encode β1, α6, and α3 subunits of heterodimer integrin, respectively, are most frequently reported in BCs and have been demonstrated to be effective BCSC markers. For example, the use of
CD133
CD133 (also known as Prominin-1) is a pentaspan and highly glycosylated transmembrane protein that defines a broad population of stem cells, including hematopoietic stem cells and endothelial progenitor cells. Although the exact role of CD133 in BC remains unclear, CD133+ BC cells assuredly display CSC-like properties. These cells showed significant resistance to DNA-damaging agents and greater capability to form tumors in NOD/SCID mice [28]. Liu [29] observed that MDA-MB-231 cells with
ALDH1
ALDH1 is a NAD(P)+-dependent enzyme that mediates the oxidation of intracellular aldehydes to carboxylic acids. Ginestier [67] found ALDH1 was a shared maker in normal and malignant mammary stem cells. Their study indicated that ALDH1 acted as an independent prognostic factor strongly associated with lower survival rate in BC patients [67]. In vitro study confirmed that CD44+CD24−ALDH1+ MDA-MB-231 and CD44+CD133−ALDH1+ MDA-MB-468 BCE cells exhibited stronger tumorigenic and metastatic capacity
CXC chemokine receptor type 4 (CXCR4)
CXCR4 is a membrane chemokine receptor. Stromal cell derived factor 1 (SDF-1, also known as CXCL12) is the only ligand for the activation of CXCR4. The SDF-1/CXCR4 signaling inherits their role in facilitating the migration of CXCR4+ BCSCs to the metastatic sites. Both CXCR4 neutralization by antibody and knockout inhibited the growth of orthotopically transplanted breast tumor and impaired their directional metastasis to lymph nodes and lung [70]. Mukherjee [31] found that non-migratory BCSCs
ABCG2
ABCG2 (also known as breast cancer resistance protein) is highly expressed in several chemoresistant BC cell lines. The trans-membrane pump protects BC cells from damage by reducing the cellular dynamic accumulation of cytotoxic drugs. The behavior is more significant in BCSCs. Compared with non-stem cells, the CD44+CD24−/low cells from MCF-7, MDA-MB-231, and SK-BR-3 BC cell lines showed higher expression of ABCG2 [37]. Similarly, the overexpression of several multidrug resistance-associated
Anthrax toxin receptor 1 (ANTXR1)
ANTXR1 is a tumor-specific endothelial marker that mediates tumor angiogenesis. The higher expression of ANTXR1 on CD44+CD24− and ALDH1+ TMD231 BC cell surface was uncovered by Chen [26]. Their work indicated that overexpression of ANTXR1 activated key genes in cell proliferation, DNA replication, and Wnt signaling pathway, conferring enhanced tumorigenic and metastatic potentials upon those BC cells [26]. By detecting ANTXR1, a subpopulation of malignant BCSCs could be sorted [26].
ANTXR1
Epithelial cell adhesion molecule (EpCAM)
EpCAM (also known as CD326 or epithelial-specific antigen, ESA) is considered as a marker for epithelial tumors and has been found to be associated with invasive BCs. Based on its role in promoting or preventing epithelial cell–cell adhesion, recent studies indicated that EpCAM plays an important role in cancer cell migration and metastasis. By detecting EpCAM+ cells, a population of circulating tumor cells (CTCs) or disseminated tumor cells (DTCs) can be separated from peripheral blood of BC
Protein C receptor (PROCR)
PROCR plays as a counterpart in maintaining the balance of tissue factor-mediated procoagulant effects through binding to coagulation proteases, such as protein C. It has been proved to be a specific CSC marker for triple negative BC. Hwang-Verslues [22] reported that PROCR+ MDA-MB-361 and MDA-MB-231 cells showed 2-fold and 9-fold increase on colony-forming efficiency, respectively, compared with PROCR− cells [22]. PROCR was demonstrated as a marker of a unique population of mouse multipotent
GD2
GD2 is a b-series ganglioside expressed mostly on the cell membrane. A small fraction of GD2+ cells identified from MDA-MB-231 cell line were capable of forming mammospheres and initiating tumors with as few as 10 cells in immunocompromised mice [80]. Most of GD2+ cells isolated from human mammary epithelial cells expressing H-Ras oncogene displayed CD44+CD24− phenotype [80]. GD3 synthase (GD3S) is involved in the biosynthesis of GD2 and associated with EMT program in BC. The expression of GD3S
Central signaling pathways sustaining BCSCs
As mentioned above, the regulating functions of BCSC surface markers cannot be abstracted from intracellular signaling. In many cases, the aberrant activation of several important signaling pathways in BC cells, as consequences of genetic mutations, epigenetic modifications, or communications with microenvironment by surface markers, generates and sustains the stem-like nature and may be a direct cause of therapeutic resistance. Thus, a better understanding of these signaling pathways may
Hh signaling pathway
Hh signaling activation gives cancer cells a survival advantage with self-renewal features as it does in organizing embryonic stem cell growth and differentiation (Fig. 2). Normally, Hh signaling is upregulated in quiescent breast stem or progenitor cells but disappears when cells undergo differentiation. Although some possible activating mutations were reported by early studies, they failed to be confirmed in larger sample sets [13]. Hence, the aberrant activation of Hh signaling in BCSCs is
Notch signaling pathway
The hyperactivation of Notch signaling cascade independent on canonical ligand induction marks a self-renewing cell population in basal BCs (Fig. 2) [46]. Notch signaling is regulated by cytokines, such as IL-6, in the tumor microenvironment. The increased IL-6 was detected in human breast tumors treated with HT. It activated cellular Notch3 signaling that can replace the ER-dependent survival mechanism and confer cancer cells a self-renewal phenotype, while blocking Notch signaling markedly
Wnt/β-catenin signaling pathway
Wnt proteins normally maintain a balance between stemness and differentiation in normal stem niches and regulate cell fate. However, constitutive Wnt/β-catenin activation due to the loss of function in negative regulators underlies the tumorigenesis in mouse mammary gland [14] (Fig. 2). The appearance of aberrant progenitor cells may account for Wnt-induced tumors, and the hyperactivated Wnt signaling in these progenitors contributes to the radioresistant phenotype in vivo [12], suggesting a
NRF2 signaling pathway
NRF2-mediated antioxidative pathway is an emerging mechanism that, at least partly, accounts for the chemo-/radio-resistant natures of cancer cells (Fig. 2). Compared with general BC cells, a much higher expression signature of NRF2 and target genes in BCSCs was uncovered, which is a collective result of proteasome reduction and p62 increase [48]. These cells benefit from the lower ROS level under CT/RT, whereas NRF2 silence retarded the formation of mammospheres and reversed the
PI3K/AKT/mTOR signaling
The sustained activation of the PI3K signaling in BCs was frequently reported in the last few years and can be basically attributed to the genetic mutations in the components of this network (Fig. 2). For example, the gain of function mutations in upstream receptor tyrosine kinases could aberrantly upregulate the activity of PI3K [16], while the loss of PTEN function was found in about 50% of BC patients [17]. Furthermore, the highly prevalent somatic mutations in PIK3CA can also increase the
Therapeutic strategies and challenges for targeting BCSCs
Currently, targeting key signaling cascades that are dysregulated in cancer cells is still dependent on the high-throughput screening of compounds and structural modification of existing drugs. Although a large number of new compounds entered into clinical trials annually and have shown satisfactory efficacy, many are withdrawn due to the poor tolerance and safety concerns. Also, some of new-generation drugs failed to abolish the immortality and stemness of CSCs that underlie the most of
Conclusion
Current evidence suggests CSC is a key target for clinical BC therapy to overcome resistance and recurrence, while findings related to surface markers and signaling network make it well-founded to develop BCSC-targeted modalities. The emergence of novel drug delivery systems can precisely remove residual BCSCs, underlying the curative promise of BCSC concept. However, the changeable and complex natures of BCSCs create several challenges. In these cases, extracorporeal tissue culture techniques
Acknowledgements
This work was supported by the St George Cancer Care Centre Research Trust Fund and the Prostate and Breast Cancer Foundation. The authors would like also acknowledge funding support from the UNSW Sydney and the China Scholarship Council.
Conflict of interest
There is no interest conflict in this research.
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