Radiation resistance in breast cancer.
Adjuvant radiotherapy continues to play a key role in the locoregional management of early breast cancer. The 2005 Oxford overview shows a clear benefit from adjuvant locoregional radiotherapy (RT) in reducing long-term 15-year breast cancer mortality . Despite this, radioresistance manifest by locoregional failure after RT remains a challenge. Even after breast-conserving surgery and postoperative whole-breast irradiation, there remains a relatively constant local failure rate of around 1% per year, at least for the first ten years . About half the patients treated with postoperative radiotherapy after wide local excision for ductal carcinoma in situ (DCIS) will develop invasive breast cancer, with its risks of distant recurrence . In addition, local control of advanced breast cancer by radiotherapy alone is rarely achieved. The mechanisms of radiation resistance in breast cancer, as in other solid tumours, are not well understood. At present there are no tests routinely applicable in clinical practice that can identify which patients will respond to radiotherapy. This article focuses on the biological basis of radioresistance, in particular on the emerging role of cancer stem cells in contributing to this phenomenon and possible strategies to overcome it.
Sensing of radiation damage
When radiation damages or disturbs DNA, signal transduction pathways are activated which slow the progression through the cell cycle and stimulate DNA repair. There are protein sensors that identify DNA damage and activate kinases. This leads to the activation of phosphorylation cascades that stop the cell cycle and repair DNA . Cell-cycle checkpoints maintain the orderly progression through the cell cycle . An important role in mammalian cells is played by the Ataxia Telangiectasia gene (ATM) and 'Ataxia Telangiectasia (ATR) Rad 3 related' in the checkpoint signalling pathways . CHK1 and CHK2 are protein kinases, activated by ATM and ATR and act as cell cycle checkpoint regulators [6,7]. The p53 gene also plays a key role in cell cycle control and programmed cell death (apoptosis) (after activation by ionising radiation). The protein of p53 acts as a substrate for ATM kinase. The complex cascade in response to DNA damage from ionising radiation is summarised in Figure 1.
[FIGURE 1 OMITTED]
In normal cells a delicate balance is kept between survival and death signals . If the amount of DNA damage exceeds the capacity to repair it, the apoptotic pathway is activated and cell death occurs. In cancers the apoptotic signalling pathway is commonly lost. There are two principal mechanisms of apoptotic signalling (intrinsic and extrinsic) (Figure 2). Cancer cells can escape apoptosis by ignoring signals from the extrinsic pathway or by resetting the balance of intracellular anti- and pro-apoptotic signals, resulting in inhibition of apoptosis . Irradiated breast and other solid cancers can thus sustain DNA damage while avoiding apoptosis. By turning off the apoptotic pathway, some cancer cells may become intrinsically radioresistant.
Mechanisms of radiation resistance
High tumour cell burden, tumour hypoxia and intrinsic/acquired resistance are three principal mechanisms of radiation resistance in breast cancer. Tumour size is inversely correlated with response to radiation. Since radiation cell killing is a random event, the higher the tumour cell burden, the higher the chance of tumour evading a lethal hit. Hypoxic cells are known to be 2.5-3-fold less sensitive to RT than normoxic cells. In addition, oxygen is needed to generate reactive oxygen species (ROS) and other free radicals thought to be necessary for the cytotoxic property of ionising radiation.
The molecular mechanisms underlying acquired radiation resistance remain to be elucidated. Numerous mechanisms are thought to contribute to intrinsic or acquired resistance, including mutated p53 , amplification of DNA repair genes, higher levels of ROS scavengers, activation of prosurvival/poor prognosis oncogenes such as EGFR and c-MET [10,11]. A variety of protein expression patterns can be induced by ionising radiation. This implies that the fate of an irradiated cell may be governed by a specific survival signalling network .Within the same tumour, that may be different subpopulations of tumour cells which respond differently to ionising radiation .
There are a number of extranuclear and intranuclear factors that may mediate radiation response. The extranuclear factors include human epidermal growth factors, insulin-like growth factor-1 receptor and the phosphatidylinositol pathway (PI-3K) and vascular endothelial growth factor. Intranuclear factors include p53, BRCA1 and BRCA2 and Bcl-2. The roles of BRCA1, BRCA2 and Bcl-2 are reviewed in detail by Jameel et al. (2004) .
Human epidermal growth factor expression
In clinical practice, overexpression of HER2 (human epidermal growth factor receptor 2) is known to be associated with an aggressive natural history of tumour growth. Recently it has been suggested  that HER2 is a DNA-damage effector gene that participates in the pro-survival signalling network and that NF[kappa]B-mediated HER2 overexpression may protect breast cancer cells from lethal effects of ionising radiation. It may be that radiation-induced genomic instability specifically activates the NF[kappa]B/HER2 pathway to increase cell survival or that it selects for radioresistant stem/progenitors with a high level of background NF[kappa]B/HER2 activity, encouraging repopulation with radioresistant clones.
[FIGURE 2 OMITTED]
Insulin-like growth factor-1 receptor
Insulin-like growth factor-1 receptor (IGF-1R), a transmembrane receptor, plays an important part in the regulation of differentiation, cell growth and apoptosis, and may play a role in breast carcinogenesis. In patients with hormone receptorpositive tumours, higher levels of IGF-1R have been found. These correlate with increased risk of recurrence and radiation resistance.
Phosphatidylinositol 3-kinase pathway
The phosphatidylinositol 3-kinase pathways play an important role in stimulating cell proliferation and stopping apoptosis. AKT is an important effector in the pathway and influences the initiation of S phase and the G2-M transition in the cell cycle. It has been shown that P1-3K/AKT activity leads to radiation resistance in breast cancer. Conversely, inhibiting P13K/AKT results in greater radiosensitivity. AKT is therefore a potential therapeutic target to increase radiosensitivity .
Overcoming intrinsic radioresistance: survivin as a target
From our understanding of the imbalance in the apoptotic pathways that may occur in breast cancer, it may be possible to inactivate pharmacological survival signals or reactivate death signals .
Survivin is a bifunctional protein involved in cell division and control of apoptosis. It is a suppressor of apoptosis and appears to play a role in resistance to ionising radiation as well as to anticancer agents. Survivin is a chromosomal passenger protein involved in metaphase, anaphase and telophase. Preclinical studies have demonstrated that downregulation of survivin function increases apoptosis and reduces tumour growth rate, and sensitises tumours both to ionising radiation and to chemotherapy. Survivin can also promote radiation resistance by supporting the survival of vascular endothelial cells. Endothelial cell apoptosis has been shown to be a major determinant of radiation response . A number of different approaches to targeting survivin are being explored, including molecular antagonists against survivin mRNA to inhibit translation, dominant negative mutants to prevent dimerisation, small-molecule antagonists to block phosphorylation and immunotherapy  (see Figure 3).
[FIGURE 3 OMITTED]
Cancer stem cells in radiation resistance
It is well recognised that the radiation sensitivity of cancer cells within the same tumour is heterogeneous . This observation is compatible with malignant tumours having a hierarchical organisation similar to normal tissue and a subpopulation of cells with the capacity to stimulate tumour growth after a sublethal treatment . There was consensus that prospectively identified cells with the capacity to generate tumours should be termed 'cancer stem cells' .
The cancer stem cell hypothesis predicts that resistance to treatment must have occurred in a CSC. It is likely that a tumour contains many subclones of CSCs. These will carry differing mutations. Exposure to ionising radiation may result in radioresistant subclones being selected and giving rise to tumour relapse. Resistance is further explained by CSCs acquiring additional mutations which increase their ability to generate proliferating progeny and to selectively promote renewal of CSCs . This might explain the observation that many tumours become resistant to therapy to which they were originally sensitive.
In many cancers including breast cancer there is evidence that only a subset of malignant cells has the capability of proliferating indefinitely and giving rise to local recurrence and distant metastases, even when the bulk of tumour cells have been eradicated . In most normal tissues there is a well regulated process by which rapidly dividing cells are lost and replenished. A limited number of stem cells give rise to daughter cells known as progenitor or transit amplifying cells. These have a limited capacity to proliferate and eventually differentiate into mature effector cells. Stem cells can also divide to produce additional stem cells to maintain the stem cell pool over the whole lifespan of the individual .
Highly tumorigenic cells whose properties are consistent with those of stem/progenitors cells have been isolated from human breast cancers . There is however, in addition, a second population of potential stem cells which may be drawn upon in the event of lethal damage to stem cells . While at low doses of radiation 4-5 clonogenic regenerating cells are recruited, this increases to 30-40 cells at higher doses . It has been speculated  that these progenitor cells are resistant to radiation and that this resistance is mediated by the beta-catenin stem cell survival pathway.
In breast cancer, CSCs were found in breast cancers which were highly enriched for CD44+CD24-low lineage-immunophenotype. Chen et al. (2006)  have studied a subpopulation of progenitor cell in the COMMA-D beta cell line from the mammary gland in pregnant mice. Using the stem cell antigen 1 (Sca1), a putative marker for mammary gland progenitors, they were able to isolate a subpopulation of Sca1+ multipotent cells that were resistant to clinically relevant 2Gy fractions. Sca1+ cells were shown to contain fewer DNA damage foci and higher levels of endogenous beta-catenin. In addition, survivin was selectively upregulated after exposure to radiation.
Diminished levels of reduced oxygen species (ROS) in cancer stem cells
It has been demonstrated that haematopoetic and central nervous system stem cells and their early progenitors have lower levels of reactive oxygen species (ROS) than their more mature derivatives. Recently it has been hypothesised  that epithelial stem cells and their cancer stem cell counterparts have the same characteristics. They found that normal mammary epithelial stem cells have lower concentration of reactive oxygen species (ROS) than their more mature progeny. Subsets of cancer stem cells in some mouse and human tumours have lower levels of ROS than non-tumorigenic cells. The relevance of this observation is that presence of reactive oxygen species is critical to the cell-killing function of ionising radiation. Cancer stem cells in these tumours acquire less DNA damage and are differentially spared compared to non-tumour cells. Lower ROS levels in cancer stem cells correlate with higher activation of ROS scavenging systems. If levels of ROS scavenging are reduced pharmacologically in cancer stem cells, their clonogenicity falls and their radiosensitivity increases.
Radiation responses of cancer stem cells
If tumour growth and regrowth are essential properties of breast cancer stem cells, it will be important to know how they respond to locoregional radiotherapy . A number of groups show the resistance of cancer stem cells in a variety of solid tumours [29,30,31,32]. One explanation for the 'shoulder' seen on the survival curve of MCF7 breast cancer cells is increased DNA repair. Diehn et al. (2009)  have shown a strong radical scavenger gene expression signature. They also observed upregulation of the Notch pathway in breast cancer stem cells via upregulation of Notch receptor ligands. Studies of endothelial cells suggest that the Notch pathway may also be involved in the radiation response .
Targeting cancer stem cells
The development of CSC-directed therapies will face significant challenges . Features of the tumour microenvironment such as tumour hypoxia will need to be addressed. Combining cytotoxic therapy with CSC-targeted agents may offer the highest chances of success. Debulking of non-tumorigenic cells with chemotherapy could be combined with CSC-directed agents. For localised and low-volume metastatic disease, radiotherapy might be combined with anti-CSC specific agents . The impact of CSC-specific therapy on normal stem cells will also need to be considered. Combining intensity-modulated RT to spare normal tissue with appropriately dosed CSC targeted therapy may be a useful avenue to explore.
Testing for radiosensitivity/resistance
At present we do not have validated markers of radiation sensitivity for breast cancer. If these could be identified, patients with radioresistant tumours could be spared adjuvant radiotherapy and its associated toxicity. From a series of patients managed by breast-conserving surgery and prospective breast irradiation, a 'wound signature' has been shown to correlated with a high risk of local recurrence . The 'wound response' gene expression signature reflects a set of genes induced or suppressed in response to bovine serum. The 'core serum response' is a powerful predictor of outcome in breast and other solid tumours . In a study of 143 axillary node-negative patients managed by breast-conserving surgery and postoperative whole-breast irradiation, a gene-expression profile could distinguish within oestrogen receptor-positive patients a group most likely to develop a local recurrence . In the current BIG 2-04 MRC/EORTC Trial of postmastectomy radiotherapy in intermediate-risk breast cancer, a tissue microarray is being established from tumours to study molecular markers of radiation response . Extensive prospective validation of these molecular signatures will be essential before they can be implemented into clinical practice.
A fuller understanding of the biological basis of radioresistance in breast cancer is needed to underpin the rationale design of therapeutic strategies to overcome it. At present there are no reliable molecular tumour markers to identify individuals likely to benefit from radiotherapy. In addition, specific drug therapies are required to target radioresistant stem cells. These should be priorities for translational research in breast cancer. Given the high proportion of patients requiring radiotherapy for breast cancer, the clinical dividends of progress in this field could be enormous.
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Edinburgh Cancer Centre, University of Edinburgh; Western General Hospital, Edinburgh, UK
Correspondence to: Ian Kunkler Western General Hospital Crewe Road Edinburgh EH4 2XU, UK (email: email@example.com)
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|Title Annotation:||Feature Article|
|Publication:||Advances in Breast Cancer|
|Date:||Sep 1, 2009|
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