In this study, we determined the relationships between CD44+CD24-/low phenotype and hypoxia markers, HIF-1α and CAIX, and classical prognostic factors in breast pathology, including clinicopathological features and the expression of immunohistochemical markers of prognostic significance (HER2 status, p53 expression and hormonal status). To the best of our knowledge this is the first study demonstrating an association between hypoxia markers and the CD44+CD24-/low phenotype in a large series of breast invasive ductal carcinomas, by immunohistochemistry.
Similar to the results of Honeth and co-workers, we found 35% of cases showing CD44+CD24-/low immunophenotype, suggesting that not all breast tumors contain cells with this phenotype, as described by Al-Hajj in 2003 [16, 2].
Since the discovery of the high capacity of generating new tumor cells by mammary CD44+CD24-/low cells, this phenotype are associated with worse survival and aggressive behavior . Sheridan and co-workers showed that CD44+CD24-/low phenotype is associated with enhanced invasive properties and elevated expression of genes involved in invasion . Further, the CD44+CD24-/low cell population was related with poor prognosis, although not in all studies . In the study of Mylona and cols., the CD44+CD24-/low phenotype was emerged as a poor prognostic indicator, within the group of grade II tumors . Furthermore, clinical studies indicate that CD44+/CD24- tumor-initiating cells express an invasive gene signature and may be associated with distant metastases [20–22]. Although the mechanisms of this aggressive behavior are not fully understood, it is now accepted that the generation of different subclones provides tools to tumorigenic cells proliferation and invasion abilities, leading to metastasis and insensitivity to classical treatments [17, 23].
Here we demonstrated that CD44+CD24-/low breast tumors show an association with HIF-1α status, but not with CAIX. Recent studies have shed light in the relationship between cancer stem cells and tumor hypoxia. There are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals such as hypoxia . Soeda and collaborators demonstrated that culture in hypoxia and activation of HIF-1α expands the sub-population of cells positive for markers associated with a more stem-like phenotype, such as CD133 and CD44 (25). A study from Pahlman and colleagues has also shown that hypoxia in neuroblastoma cells can alter gene expression to that of a more immature phenotype.26 There are recently published data demonstrating this critical role of hypoxia in maintaining the stem-like fraction of cancer cells [27, 4, 28].
Low oxygen levels promote maintenance of embryonic stem cell pluripotent potential and block differentiation . In a recent study, Bar and co-workers showed that gliobastoma neurosphere cell lines and freshly resected tumors respond to hypoxia by an increase in cancer stem cell marker CD133 expression. These neurospheres also exhibited a significant increase in clonogenic capacity, after 12 and 24 hours of hypoxia. According to the authors, HIF-1α alone can mediate the majority of hypoxic effects in gliobastoma neurospheres, indicating a pivotal role of HIF-1α in clonal expansion of cancer stem cells .
Further findings indicate that more than co-acting in cancer stem cell maintenance, hypoxia could drive the maintenance of a cancer stem cell niche, with a strong participation of HIF-1α. Heddleston and colleagues showed that non-stem glioma cells cultured under hypoxic conditions had twice the rate of formation of neurospheres, compared to cells in normoxia, suggesting that a hypoxic microenvironment plays a pivotal role in promoting and maintaining the ability of self-renew in stem-like cells, and even conferring self-renewal capability to non-stem cell population .
Our finding that HIF-1α is related to CD44+CD24-/low immunphenotype in breast ductal carcinomas could indicate that a maintained hypoxia state in breast cancer could facilitate the maintenance of cancer stem cells in these tumors, corroborating the finding that CD44+CD24-/low positive tumors have a worse prognosis when compared to non- CD44+CD24-/low tumors. The association between negativity to CD44+CD24-/low immunophenotype and negativity to HIF-1α and CAIX expression could, at least in part, explain the better survival of this group of patients, and also the worst prognosis associates with CD44+CD24-/low cases.
We also found an association between CD44+CD24-/low phenotype and HER-2 status. Patients lacking CD44+CD24-/low phenotype showed negativity to HER-2 immunohistochemycal expression. Recently, Oliveras-Ferraros and co-workers had demonstrated that CD44+CD24-/low cells could be important in de novo resistance to direct HER inhibitors such as trastuzumab (Herceptin©), a HER-2 directed antibody, and other studies suggest that the clinical efficacy of trastuzumab may relate to its ability to directly target the CSC population in HER-2-amplified tumors [30, 22, 23].
In fact HER-2 positive tumors constitute a group with a worse prognosis, when compared to tumors lacking HER-2 expression. The relationship between non- CD44+CD24-/low tumors and negativity to HER-2 could explain the better prognosis found in this group of patients.