Neoadjuvant chemotherapy (NAC) is the standard care for locally advanced breast cancer and is used increasingly for large breast cancer with the intention of tumor down-staging so that breast-conserving surgery could be considered as a surgical option [35
]. IMPC of the breast is a distinct and aggressive variant of breast cancer that shows marked lymphotropism, extensive axillary lymph node involvement at diagnosis, and frequent local recurrence and distant metastases at follow up. In addition, Alvarado-Cabrero et al. have recently reported that none of the IMPC of a case series of locally advanced breast cancers treated by NAC did respond to treatment [7
]. To our knowledge, no data exist on the molecular basis of the lack of response to chemotherapy in IMPC of the breast.
In the present study, using a 3-D model that mimics the small papillae typical of the IMPC growth pattern, we demonstrated that HIF-1 activation may be related to the drug resistance described in this tumor. We decided to use the ER-positive MCF7 cancer cells because the IMPC are, in most cases, ER-positive breast cancers.
We first observed that HIF-1α is translocated into the nucleus and is able to bind a HRE both when MCF7 cells are cultured as 3-D spheroids or as monolayers under hypoxic conditions. In contrast, MCF7 cell monolayers under normoxic conditions exhibit a minimal activation of HIF-1. We also showed that expression of PDK1, PGK1
, and VEGF
, well-known HIF-1 target genes, was increased in MCF7 3-D spheroids and MCF7 hypoxic cells compared to MCF7 control cells. This observation suggests that in vitro MCF7 3-D spheroid formation may mimic hypoxic conditions and that the activation of HIF-1 in MCF7 3-D spheroids may be an adaptation to hypoxia. This result is in agreement with previous reports showing that cells cultured as 3D-spheroids are associated with a microenvironment characterized by gradients in O2
, pH, and nutrients, which simulates the architecture of tumors better than the 2-D or monolayer cell culture model [27
It has been postulated that hypoxia in solid tumors dramatically decreases the chemosensitivity of tumor cells and that experimental hypoxia promotes drug resistance to anticancer agents in a variety of cell lines [12
]. In particular, Comerford et al. revealed that hypoxia induces MDR-1
gene expression and concomitant functional expression of its product Pgp in cells grown as monolayers under hypoxic conditions and in cells grown as multicellular spheroids. Moreover, they identified a functional HIF-1 binding site within the MDR-1
gene promoter [16
]. Later on other groups confirmed that the activation of HIF-1 in tumor cells is associated with an increase in Pgp expression [17
]. These findings are consistent with the present results reporting that binding of HIF-1α to MDR-1
gene promoter is higher in MCF7 3-D spheroids and in MCF7 hypoxic cells than in MCF7 control cells. In addition, we observed an increased expression of Pgp on the surface of cells obtained from 3-D spheroids and from monolayers under hypoxic conditions. This increase is abolished both by the incubation of cells with YC-1, which induces a decrease in HIF-1α accumulation by down-regulating HIF-1α mRNA translation [29
], and by the transfection of cells with specific siRNA for HIF-1α. Our results suggest that in both MCF7 3-D spheroids and MCF7 hypoxic cells HIF-1 is activated and is recruited to participate to the transcriptional activity of MDR-1
gene, and that the increase of Pgp surface-expression is associated with this activation. It is noteworthy that Pgp is a predominant membrane transporter associated with chemotherapy resistance [39
]. Here we demonstrated that MCF7 3-D spheroids and MCF7 hypoxic cells accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as a monolayer under normoxic conditions. The inhibition of HIF-1α, either by incubating cells with YC-1 or by transfecting cells with specific siRNA for HIF-1α, increases the doxorubicin accumulation in both MCF7 3-D spheroids and MCF7 hypoxic cells to a level comparable to the one of MCF7 control cells, confirming that HIF-1 activation induces doxorubicin resistance. It has been previously demonstrated that doxorubicin induces apoptotic cell death in cancer cell lines [31
], even though doxorubicin has been also reported to induce senescence in tumour cells [40
]. In the present study, the percentage of annexin V-positive cells and the caspase-9 activity of MCF7 control cells exposed to doxorubicin are low. However, we did not observe in MCF7 control cells enlarged and flattened cells, the predominant morphologic change associated with the onset of drug-induced senescence [41
], and we observed significant differences in percentage of apoptotic cells and caspase-9 activity in the following couples: 1) MCF7 control cells and MCF7 3-D spheroids in the absence of YC-1; 2) MCF7 3-D spheroids in the absence of YC-1 and MCF7 3-D spheroids in the presence of YC-1. Moreover, we restricted our interest to doxorubicin because this drug is widely used to treat ER + breast cancer in clinical setting.
Recently, Hung et al. demonstrated that YC-1 is able to modulate the transport function of Pgp and thus to chemosensitize hypoxic tumor cells via the nitric oxide (NO)- cyclic GMP (cGMP)- cGMP-dependent protein kinase (PKG)- extracellular signal-regulated kinase signaling pathway through noncompetitive inhibition [42
]. Previous studies have shown that various NO mimetics, including 8-bromo-cGMP, attenuate hypoxia-induced resistance to several chemotherapeutic agents including doxorubicin, suggesting that part of the mechanism of hypoxia-induced drug resistance in cancer cells is the suppression of endogenous NO production [43
]. Moreover, Frederiksen et al. demonstrated that chemosensitization of hypoxic tumor cells by NO mimetics requires activation of soluble guanylyl cyclase, generation of cGMP, and activation of PKG [45
]. Our research group has also reported that a doxorubicin-resistant (HT29-dx) cell population obtained from epithelial colon cell line HT29 accumulates less intracellular doxorubicin, is less sensitive to the cytotoxic effects of the drug, overexpresses Pgp, and exhibits a lower NO production. The resistance to doxorubicin is reversed by inducers of NO synthesis [46
]. In the present study, even if we cannot exclude that YC-1 activates the NO-cGMP-PKG signaling pathway, the inhibition of HIF-1α by transfecting cells with specific siRNA for HIF-1α seems to confirm the previously reported inhibitory action of YC-1 on HIF-1α mRNA translation [29
We demonstrated that the presence of verapamil restores the intracellular doxorubicin content observed in MCF7 3-D spheroids to a level comparable to the one of MCF7 control cells, suggesting that MCF7 3-D spheroid formation induces a resistance to doxorubicin, at least in part, via Pgp. However, verapamil, a commonly used first-generation Pgp inhibitor, is also able to inhibit other ABC transporters such as MDR3 and some multidrug resistance-associated proteins (MRPs) [47
]. Verapamil is nevertheless not able to inhibit MRP1 and the breast cancer resistance protein, two other ABC transporters associated with a MDR phenotype in cancer cells [48
] and expressed in MCF7 cells (data not shown), suggesting that Pgp may have an important role in the MDR phenotype in MCF7 3-D spheroids.
Finally to confirm that our 3D-culture model produces cell clusters truly similar to IMPC, we evaluated whether the inside-out growth pattern of IMPC was reproduced in vitro [49
]. The peculiar architecture of IMPC of the breast has been attributed to a rotation of cell polarization so that the surface of the tumor cells that faces the stroma acquires apical secretory properties and detaches from the stroma. This was shown first by electron microscopy [50
] and then confirmed by immunohistochemical staining for MUC1 [33
]. MUC1 is a glycoprotein normally located in the apical cell surface of normal glandular epithelium. In conventional carcinomas MUC1 is largely apical, with intracytoplasmic or intercellular distribution [51
]. In IMPC, MUC1 has a highly aberrant expression being localized predominantly in the stroma-facing surface of the cells [33
]. In this study, we obtained in vitro 3-D spheroids by treating MCF7 cells with human neutrophil elastase, as described previously [21
]. We demonstrated that the morphological similarity observed between MCF7 3-D spheroids and IMPC of the breast corresponds to a similar distribution of MUC1. Indeed, in MCF7 3-D spheroids MUC1 is expressed along the outer cell surface of the clusters. The mechanism of neutrophil elastase-induced formation of MCF7 3-D spheroids with inverted orientation of epithelial polarity is yet unknown. Moreover, we showed that MCF7 3-D spheroids express markers of biological aggressiveness such as CD44 and p53 similarly to IMPC.
We showed that in breast biopsy from patients with IMPC HIF-1α is expressed in the nucleus of the cells similarly to MCF7 3-D spheroids. This result is in agreement with previous reports showing that ER + breast cancers express strong levels of HIF-1α [52
] and its expression is correlated with diagnostic and prognostic indicators for early relapse and metastatic disease [53