In this study we analyzed the relationships among hypoxia-inducible proteins (i.e., HIF-1α, Glut-1, leptin, and ObR) and other biomarkers (i.e., estrogen receptors and Ki-67) in primary and metastatic breast cancer. Our second goal was to evaluate potential influence of preoperative chemotherapy on associations among studied proteins. The major findings of our study can be summarized as follows: 1) HIF-1α and Glut-1 are positively correlated in primary tumors without preoperative chemotherapy; 2) HIF-1α positively correlate with the leptin system in primary and metastatic breast cancer without preoperative chemotherapy; 3) the leptin system is not associated with Glut-1 expression in all studied groups; 4) Glut-1 correlates negatively with ERα in primary and metastatic tumors, regardless of preoperative chemotherapy; 5) hypoxia markers correlate positively with ERβ expression in primary tumors, especially in the group without preoperative therapy; 6) Glut-1 expression is positively associated with Ki-67 in primary tumors, while in lymph node metastases, a trend toward positive correlation between these proteins is found in the group without therapy; 7) preoperative chemotherapy influences the associations between HIF-1α and leptin in primary and metastatic tumors, HIF-1α and ObR in metastatic tumors, HIF-1α and Glut-1 in primary tumors; Glut-1 and ERβ in primary tumors; Glut-1 and Ki-67 in primary and metastatic tumors.
Hypoxia in solid tumors is associated with the accumulation of HIF-1α and activation of HIF-1-dependent transcription of genes regulating cell motility, invasion, and angiogenesis [14, 41]. Bos et al. (2001, 2003) reported that HIF-1α overexpression was associated with more aggressive breast cancer. We speculate that HIF-1 could also improve glucose consumption in hypoxic cancer cells by the stimulation of glucose transporter Glut-1 expression [12, 42]. Our results suggest that hypoxic conditions resulted in coordinated upregulation of HIF-1α and Glut-1 in primary tumors. It is consistent with results of Li et al. (2006) who showed that knockdown of HIF-1α in MCF-7 breast cancer cells attenuated the expression of Glut-1 mRNA and other genes . Additionally, the HIF-1α knockdown cells displayed increased sensitivity to chemotherapeutic agents . Interestingly, we noted that the association between HIF-1α and Glut-1 was lost in lymph node metastases as well as in tumors after preoperative chemotherapy. Potentially, this could be related to downregulation of HIF-1 coregulators, such as p300 , due to neoadjuvant therapy or genetic rearrangements in metastatic cells , which could result in deficient Glut-1 gene transcription [46, 47].
The associations between HIF-1α and the leptin system identified by us in this study confirm the results obtained by Cascio et al. (2008), Bartella et al. (2008) and Garofalo et al. (2006) who demonstrated that physiologic hypoxia and/or accumulation of HIF-1α due to hypoxia-mimicking conditions or stabilization of HIF-1a by growth factors can stimulate leptin and/or ObR expression in breast cancer cells [34, 36, 37].
According to our knowledge, the present analysis is the first to reveal a positive correlation between HIF-1α and leptin, as well as between HIF-1α and ObR not only in primary tumors, but also in metastasis [31, 33, 34, 36–38]. We reported analogous associations in human colorectal cancer . Cumulatively, present and previous data suggest the involvement of tissue hypoxia in the stimulation of leptin and ObR expression in human cancers [28, 30, 34]. The overexpression of the leptin system could lead to leptin-enhanced tumor growth and progression under hypoxic conditions.
One of the features of breast cancer progression is the development of resistance to hormonal therapy. The possible mechanism of this phenomenon could be related to differential ER expression in metastatic and primary sites, as described by us before . Our present study suggests that tumor malignancy might correlate with loss of ERα expression. Indeed, hypoxia could induce ERα downregulation via proteasome-dependent pathway [48, 49]. Cho et al. (2005) suggested that ERα downregulation under hypoxic conditions in human breast cancer involves protein interactions between ERα and HIF-1α .
Negative correlation between Glut-1 and ERα found in this study suggests that loss of ERα in breast cancer is associated with overexpression of Glut-1, which could facilitate proliferation, survival and possibly progression and dedifferentiation of cancer cells [51, 52].
In contrast to ERα, we noted a positive correlation between ERβ and hypoxia markers HIF-1α, Glut-1. Our observations are consistent with results of Cordadini et al. (2004), who reported that hypoxic conditions upregulated ERβ protein levels in breast cancer cells . Moreover, sequence analysis of the ERβ promoter region contains specific sequence for HRE, which might explain coexpression of ERβ and HIF-1α.
Although preoperative chemotherapy is an integral part of management of patients with advanced breast cancer, its impact on cancer biomarkers and their interrelationships in primary and metastatic tumors is poorly recognized. Our study clearly suggest that preoperative chemotherapy alters the expression of and relationships among several hypoxia-related proteins, specifically, HIF-1α and leptin in primary and metastatic tumors, HIF-1α and ObR in metastatic tumors, HIF-1α and Glut-1 in primary tumors; Glut-1 and ERβ in primary tumors; Glut-1 and Ki-67 in primary and metastatic tumors. These data are reminiscent of previous observations pointing to differential expression of ERα, ERβ and Ki-67 expression in metastatic breast cancer versus primary cancer, and significant influence of preoperative chemotherapy on these biomarkers [40, 43, 54–56].