In summarizing our study, CD133 was exclusively expressed in cancer cells compared to stromal and immune cells and was associated with other CSC markers (CD24, NOTCH1, DLL1, and ALDH1A1), as well as enriched WNT/β-Catenin, Hedgehog, and NOTCH signaling, validating CD133 as a CSC marker. We found that the expression of the cancer stem cell marker CD133 is associated with reduced cell proliferation and DNA repair, yet heightened inflammation, and is linked to a more favorable outcomes after NAC and improved survival among ER-positive/HER2-negative BC patients.
Based on the fact that the CSCs are less proliferative than other types of cells in the tumor, we expected the expression of the CSC marker CD133 to be related to less cell proliferation. However, Joseph et al. reported that CD133 is associated with greater cell proliferation, less response to NAC, and worse prognosis in invasive BC [17]. Our data was consistent with our expectation and contradicted Joseph et al.’s report, which analyzed invasive BC as a whole, as opposed to our study that specifically investigated the ER+/HER2- subtype based on the understanding that biology and characteristics are significantly different by subtypes. It may be worth noting that CD133 protein expression evaluated by flow cytometry did not correlate with its mRNA expression level [54].
Our team, alongside other investigators, has reported an association between DNA repair enhancement and cell proliferation [18, 52]. The same trend has been shown by Oshi et al. in hepatocellular carcinoma [45], who found that enhanced DNA repair was associated with a worse prognosis and more cell proliferation but not with the fraction of immune cell infiltration nor immune response. Consistently, high expressions of RAD51 [18] or BRCA2 [52], both of which play a critical part in DNA repair, were associated with increased cell proliferation and aggressive biology in BC. Given that CD133-high BC was associated with less cell proliferation, its association with less DNA repair may explain its mechanism. On the other hand, Cheah et al. reported that CD133-marked putative CSCs correlated with proficient mismatch repair [55], thus multiple mechanisms may be involved in the relationship between CD133 expression and DNA repair.
We also found that inflammation and immune response were enriched in CD133-high TME. The number of many types of infiltrating cells in TME were not significantly different between high and low CD133 tumors and, interestingly, some types of cells were negatively correlated with CD133 expression. However, cytolytic activity, which represents the overall activity of the immune cells and thus cancer immunity, was significantly and positively correlated with high CD133 expression. It remains unclear precisely how and, after all, whether low DNA repair leads to high inflammation in the TME. Several previous studies reported that in several cell lines and cancer types, low DNA repair led to a higher neoantigen load, therefore high immunogenicity, and, as a result, more lymphocytes infiltration and richer inflammation [38, 56]. Nevertheless, while we observed slightly higher silent and non-silent mutation rates in CD133-low tumors, no discernible difference was noted in SNV neoantigens and indel neoantigens based on CD133 expression. This observation diminishes the persuasiveness of that explanation in our study. However, there are still several possible mechanisms that impaired DNA repair results in richer inflammation in TME, although not in higher loads of neoantigens. One is through the accumulation of DNA damage and subsequent activation of several signaling pathways such as the ATM/ATR pathway and the DNA/PK pathway, which can lead to the activation of NFκB and other pro-inflammatory transcription factors that induce the production of pro-inflammatory cytokines, chemokines, and growth factors by cancer cells and surrounding immune cells [57]. This hypothesis is further supported by the fact that TNFα signaling via NFκB is enriched in CD133-high tumors in our study (Fig. 5C), which is also known to enrich inflammation [58]. Another possible explanation is that impaired DNA repair results in the accumulation of damaged or misfolded proteins in the endoplasmic reticulum of cancer cells, leading to endoplasmic reticulum stress and activation of the unfolded protein response (UPR). The UPR can also activate pro-inflammatory pathways, leading to the production of pro-inflammatory cytokines and chemokines [59–60], although several previous studies suggest that the chronic activation of UPR is considered a mechanism of tumor progression [61–63], going against better DFS and OS observed in our study, which may be due to the difference in cohorts.
Finally, and most importantly, we found that CD133-high BC carried a better survival outcome. We cannot help but speculate that while CD133-high tumors have a poor prognosis, as previous studies suggest accordingly with the cancer stem cell concept that involves self-renewal, differentiation, and the initiation of tumorigenesis, CD133-low tumors may carry even worse prognosis due to their ability to repair DNA, more cell proliferation, decreased immunogenicity, hence less response to NAC and worse survival outcome. The correlation between inflammation and pCR in invasive BC has been proposed by Hatzis et al. [36]. Furthermore, less cell proliferation in CD133-high BC may explain better prognosis, going along with some prior findings that showed an association between more expression of genes related to proliferation such as G2M [26–27], E2F [23, 25], and MYC [64] and worse prognosis in ER+/HER2- BC. In summary, the association of elevated CD133 expression in breast cancer cells with diminished DNA repair, improved response to NAC, and enhanced survival underscores CD133's potential role as a marker for predicting the treatment response in ER+/HER2- subtype BC.
Our method is subject to certain limitations inherent in the essentially retrospective nature of this study. Firstly, the utilization of patient sample data from a public domain means the analysis relies on information that had previously been cataloged, resulting in limited granularity. Secondly, the origin of the sample within the bulk tumor may vary among patients, even though the spatial relationship of CSCs in the bulk tumor may be of importance. It has been indicated that CSCs at the periphery of the bulk tumor may not have been sampled [65], although CD133 is known to be particularly upregulated in low O2 tissues [66]. These biases may have resulted in an underrepresentation of the full array and functionality of the CSCs.