The role of radiotherapy-resistant stem cells in breast cancer recurrence

Although the effectiveness of anti cancer therapies has improved dramatically, breast cancer (BC) remains the second leading cause for cancer-related death among women in the western world [1]. The observed improvement in the survival rate and remission for primary tumors are essentially related to the advances in early diagnosis with more sensitive imaging technology [2,3]. However, the death rate remains unacceptably high for BC patients due to hard-to-treat metastatic and recurrent tumors, necessitating new, effective approaches and anticancer agents. Accumulating evidence suggests that cancer stem cells (CSCs), which are present in many cancers including BC, mediate tumor metastasis and contribute to relapse due to their resistance to current conventional therapies [4,5]. These unique cells possess stem cell-like characteristics, such as the capacity of self-renewal, which makes the tumor capable of regenerating its entire bulk. CSCs are resistant to proapoptotic factors, rendering them a formidable adversary to anticancer agents. In part, this is related to their quiescence capacity, which holds them in a standby mode in their niche microenvironment, sheltering them from radiation and other anticancer agents, since these agents are effective only on highly proliferative cells [6,7]. In addition to their powerful DNA repair machinery, breast CSCs (BCSCs) express ALDH, which is suspected to play a part in their death-resistance phenotype by being involved with cell detoxification machinery [8,9]. Thus, BCSCs represent a major challenge in the battle against BC, and an opportunity to develop more effective target to treat metastatic lesions.

Radiotherapy (RT), a recognized treatment modality for BC patients [10,11], is also considered appropriate for patients with high risk of recurrence [12]. Although substantial benefits are achievable with this treatment, especially for ductal carcinoma and early invasive cancer, advanced invasive tumors can develop radioresistance, and the molecular mechanisms involved are yet unknown. Recently, tumor radio-resistance has been linked to the activation of NF-κB, a ubiquitous transcriptional factor in mammalian cells [13]. The induction of NF-κB is associated with the activation of an array of antiapoptotic genes, such as MnSOD and MKP1, which scavenge damaging free radicals and inhibit the apoptosis signaling pathway (JNK) [14,15]. Interestingly, the NF-κB-regulated genes act partially on the apoptosis mechanism mediated by mitochondria [16,17], since some of those proteins are detected in the mitochondria in response to irradiation. These data suggest that mitochondria metabolism is involved in the development of radioresistant CSCs. Thus, tumor cells, especially BCSCs, are able to adapt DNA-damaging anticancer agents (e.g., RT and chemotherapy) via an NF-κB-controlled prosurvival network, which contributes to the death resistance and aggressiveness of recurrent BC. Therefore, the combination of the intrinsic properties of CSCs and their radiation-adaptive responses due to NF-κB-regulated genes should be considered targets for the elimination of CSCs and subsequent prevention of tumor recurrence. However, this cannot be achieved until reliable markers for these resistant BCSCs are identified.

BCSCs are recognized by specific biomarkers that are difficult to distinguish from normal somatic stem cells. Creighton et al. found that a residual BC cell population that survives after chemo therapy is enriched by cells with both BCSC and mesenchymal features [18]. They defined a gene expression signature common to both CD44⁺/CD24^−/low and epithelial–mesenchymal transition-associated gene expression. Recently, HER2, a tyrosine kinase receptor involved in BC, has been considered a reliable marker for CSCs [19,20]. HER2 is a proto-oncogene that encodes a transmembrane protein in various tissues [21]. Probably owing to its high gene dosage, HER2 overexpression in BC correlates with the aggressiveness and high risk of tumor relapse and is an indicator for poor prognosis [22]. Although no agonist has been identified, the activation of HER2 is associated with highly invasive tumors. HER2 activation can be triggered by homo- or hetero-dimerization with other members of the tyrosine kinase receptor family. The development of neutralizing antibodies against the transmembrane domain of the receptor has dramatically improved the outcome of HER2⁺ BC patients identified by FISH and histochemical analyses [23]. The usage of Herceptin®/trastuzumab as an adjuvant treatment sensitizes BC cells to both RT and chemotherapy, and the survival rate is substantially extended for HER2⁺ patients [24]. However, Herceptin is mainly applied to patients with an amplified HER2 gene [25]. Jones and Buzdar suggest that the efficacy of anti-HER2 therapy is related to the targeting of BCSCs in HER2⁺ patients, which accounts for 28% of BC patients [26]. Supporting this hypothesis is the capacity of HER2 overexpression to promote the enrichment of stem cells in both normal and malignant cells, even HER2⁻ BC cells [19,20]. The remaining question is whether the enrichment of HER2⁺ BCSCs was related to the death or low expansion rate of HER2⁻ BCSCs, or to the transformation of HER2⁻ CSCs to HER2⁺ cells, especially during the process of chemotherapy and RT. The other key question is whether the role of HER2 is limited only to patients in whom the HER2 gene is amplified. Guo et al. reported that HER2 gene expression could be induced in response to radiation [27], and Magnifico et al. showed that, compared with parental BC cells, HER2 is highly expressed in CSCs, rendering them sensitive to Herceptin. However, the enhanced HER2 protein level may not be only related to HER2 gene dosage, but also to the activation of HER2 gene expression at the transcriptional level via the NF-κB signaling pathway [28,29]. Furthermore, the HER2 protein level is also linked to post-transcriptional modifications [30]. Therefore, HER2 expression in BC appears to be a dynamic process and depends on intrinsic (genetic inheritance) and/or extrinsic factors (RT, chemotherapy and cytokines); such changes could alter the dynamics of the CSC subpopulation pool size during or after anti cancer treatment. Taken together, these data suggest that, besides the gene dosage in a given tumor cell, HER2 gene transcription and post-translational modification are involved in the adaptive resistance of tumors to RT and RT/chemotherapies. In these cases, NF-κB-mediated HER2 transcription may account for the enrichment of the CSC population in the tumors that are resistant to RT and chemotherapy.

Therefore, HER2 may serve as a biomarker for BCSCs, and signaling pathways involved in its regulation and/or mediating its effects should be taken into account when targeting CSCs [26]. RT has been shown to promote the enrichment of CSCs through the induction of HER2 expression [19], and this is related to the capacity of radiation-induced NF-κB to bind and activate the HER2 gene promoter [28]. However, it has been noticed that HER2⁺ BCSCs can be detected not only in HER2⁺ tumor cells, but also in HER2^−/low BC [19]. These results highlight an effect of irradiation in the repopulation of BCSCs due to changes in HER2 expression status. In support of this, Malik et al. found that the bone micro-environment affects HER2 expression in MCF7 cells (HER2^−/low), suggesting that HER2 status is also related to the host microenvironment tissue, where metastatic tumor growth occur [31]. Thus, due to the critical functions of HER2 in the promotion of CSCs, the dynamic patterns of HER2 levels should be taken into account when designing therapeutic protocols, even for BC with HER2^−/low status. Furthermore, the presence of HER2⁺ BCSCs in HER2^−/low BC may explain the benefit of anti-HER2 therapy observed in patients for whom HER2 gene amplification was not detected. Elucidating the radioresistance mechanisms of BCSCs, such as the HER2–STAT3 pathway [19,32], will provide additional new insights into factors involved in the formation of radioresistant BCSCs.

Conclusion

We conclude that BCSCs are responsible, at least in part, for the radioresistant phenotype of recurrent and metastatic lesions of BC. Targeting chemo/radio-resistant BCSCs is a potentially effective approach to obtain total remission and further enhance the cure rate for BC patients. The identification of HER2⁺ BCSCs in HER2⁻ tumors will enable us to discover new biomarkers in order to trace therapy-resistant BCSCs and to target/kill potential early-stage metastatic lesions. Thus, not only should the HER2 gene dosage detected in a given tumor biopsy be taken into count when designing a proper treatment plan, but also the dynamic pattern of HER2 protein expression, as well as HER2-associated signaling pathways. In this regard, NF-κB-mediated HER2 over expression in tumor cells, progression and repopulation due to the enhancement of radioresistant BCSCs should be investigated further as potential effective targets to treat recurrent BC.

“Cancer stem cells are resistant to proapoptotic factors, rendering them a formidable adversary to anticancer agents.”

“Accumulating evidence suggests that cancer stem cells ... mediate tumor metastasis and contribute to relapse due to their resistance to current conventional therapies.”

“Elucidating the radioresistance mechanisms of breast cancer stem cells ... will provide additional new insights into factors involved in the formation of radioresistant breast cancer stem cells.”

Biography

Cheikh Menaa

Jian Jian Li