Introduction
Extracellular acidosis is a frequent feature of the microenvironment in solid tumors, and acidosis is considered one of the major selective forces that promote evolution of aggressive and drug-resistant tumor clones [
1]. Enhanced glycolysis with lactacidosis is a key contributor to reduced extracellular pH (pH
e) in tumors [
2].
In vivo measurements have revealed pH
e values of 6.5 to 6.9 in human breast cancer and other malignant tumors compared to normal tissue pH
e values of 7.2 to 7.4 [
3,
4]. Cancer cell glycolysis may be anaerobic as a consequence of hypoxia (the Pasteur effect) or aerobic due to metabolic reprogramming even under normoxic conditions (the Warburg effect) [
5]. In addition, glycolysis in cancer-associated fibroblasts may contribute to extracellular acidosis in the tumor microenvironment [
6]. Importantly, extracellular acidosis of malignant tumors potentiates cancer progression by facilitating tumor invasion [
7,
8], suppressing immune responses [
9] and promoting metastasis in mouse models [
10,
11]. Elevated lactic acid secretion and acidosis were also associated with higher incidence of metastases in various human cancers [
12,
13]. However, the molecular mechanisms underlying selection for more aggressive cancer clones during acidosis remain incompletely understood and may vary between cancer types.
In breast cancer, prolactin has been implicated as a tumor promoter based on experimental studies in rodents [
14,
15] and the association between elevated circulating prolactin levels and increased risk of developing breast cancer [
16]. Prolactin sustains nuclear tyrosine phosphorylated Stat5 (Nuc-pYStat5) and supports survival and expansion of differentiated luminal breast epithelial cells [
17,
18] and maintains their sensitivity to cell death [
19]. In breast cancer cell lines, experimental activation of Stat5 promotes differentiation, inhibits invasive characteristics [
20‐
22], and blocks progesterone-induced emergence of a drug-resistant CK5-positive cell population [
23] with tumor-initiating characteristics [
24‐
26]. In clinical breast cancer specimens, loss of Nuc-pYStat5 is associated with poor prognosis and increased risk of tamoxifen resistance [
27‐
30]. Thus, a dual role of prolactin-Stat5 signaling in breast cancer has been proposed, wherein initial pathway activation promotes cell survival and tumor formation, whereas differentiation-promoting effects of prolactin-Stat5 signaling may support homotypic adhesion and suppress subsequent invasive behavior and progression [
31]. However, little is known about the molecular causes for frequent loss of Stat5 tyrosine phosphorylation in human breast cancer.
Intriguingly, surface plasmon resonance studies have shown that prolactin interaction with its receptor is disrupted at pH of 6.0 or lower [
32]. Such low pH occurs in early endosomes and in prolactin secretory vesicles of pituitary lactotrophs and may facilitate recycling of prolactin receptors and reversible prolactin aggregation, respectively [
32]. Although pH
e lower than 6.5 rarely occurs in extracellular space of solid tumors, it has remained unclear, based on limited
in vitro experiments [
32‐
34], whether such moderate extracellular acidosis of the microenvironment of breast cancer affects prolactin signaling. Based on
in vitro and
in vivo experimental approaches and extensive quantitative
in situ analyses of human breast cancer specimens, we now demonstrate that prolactin activation of prolactin receptors is selectively disrupted even at mildly acidic pH
e of 6.8. The new observations identify acidosis as a significant contributor to loss of Nuc-pYStat5 in clinical breast cancer specimens, and implicate acidosis-induced prolactin resistance as a previously unrecognized mechanism by which breast cancer cells may evade homeostatic control.
Discussion
The present study supports the novel pathophysiological concept that extracellular acidosis within the microenvironment of breast cancer potently and selectively disrupts prolactin receptor signaling, including Stat5 activation. Previous analyses of more than 2,000 cases revealed that loss of nuclear translocated and tyrosine phosphorylated Stat5 (Nuc-pYStat5) occurs frequently in breast cancer, and correlates with disease progression, poor prognosis, and increased risk of resistance to endocrine therapy [
27‐
30]. Intratumoral acidosis is a previously unrecognized factor contributing to loss of prolactin-induced Nuc-pYStat5 human breast cancer, and implicates acidosis-associated prolactin resistance as a novel mechanism by which breast cancer cells escape pro-differentiation and invasion-suppressive effects of prolactin.
The pathophysiological relevance of potent and reversible acidosis-disruption of prolactin signaling in breast cancer is supported by extensive experimental evidence and correlative studies in archival human breast cancer specimens provided clinical relevance. Indeed, we observed mutually exclusive expression patterns of Nuc-pYStat5, a marker of prolactin receptor activation, and elevated levels of GLUT1, a marker of increased glycolysis and associated lactacidosis. Quantitative multiplexed immunofluorescence analyses specifically revealed that positive GLUT1 expression (gain-of-function) was associated with low levels of Nuc-pYStat5 (loss-of-function) in malignant breast tumors at three different scales: at the global tumor level, regionally within tumors, and at the cellular level. This is consistent with global and regional acidosis within malignant breast tumors. However, a substantial number of tumors, tumor regions, or tumor cells were negative for both GLUT1 and Nuc-pYStat5, indicating that not all tumor-associated absence of Stat5 signaling is explainable by GLUT1-associated acidosis in breast carcinoma cells. For instance, acidosis may in some cases be caused by increased glycolysis within stromal fibroblasts [
6], which is not correlated with epithelial GLUT1 staining. Furthermore, alternative mechanisms likely lead to loss of Nuc-pYStat5 in human breast cancer, such as inhibition of prolactin-Stat5 signaling by the tyrosine phosphatase PTP1B through inhibition of the Jak2 tyrosine kinase [
46].
Aerobic glycolysis in carcinoma cells is frequently associated with activated oncogenes, including Src, Myc, AKT/mTOR pathway and mutation of tumor suppressors such as p53 [
47]. In these cases, the entire tumor typically displays glycolytic metabolism regardless of oxygenation status. In fact, we observed that more than half of the GLUT1-positive human breast cancer specimens displayed generally homogenous GLUT1 staining throughout the tumor and were essentially negative for Nuc-pYStat5. Alternatively, rapidly proliferating tumors may exhibit regional hypoxia with resulting focal or regional hypoxia-induced glycolysis and acidosis. Indeed, heterogeneous GLUT1 staining in breast tumors was also commonly detected, including specimens with GLUT1-positive foci surrounding necrotic regions suggestive of local hypoxia. More recently, paracrine hepatocyte growth factor from cancer-associated fibroblasts was shown to promote GLUT1 expression and the Warburg effect in cancer [
48]. Regardless of the mechanisms underlying increased regional glucose metabolism and extracellular acidosis, carcinoma cells positive for Nuc-pYStat5 were absent in tumor regions where carcinoma cells displayed elevated GLUT1. Experimental evidence for acidosis-induced suppression of prolactin signaling in breast cancer was extended from cell lines in two-dimensional cultures to human breast cancer xenotransplants in mice
in vivo and to multilayered three-dimensional spheroid cultures, experimental conditions that better mimic local acidosis within the patient tumor microenvironment. In fact, T47D xenotransplant tumor regions expressing high GLUT1 were resistant to exogenous prolactin despite retaining prolactin receptor and Stat5 expression. Furthermore, in three-dimensional spheroids of T47D cells extracellular alkalinization alone rapidly reversed acidosis-disrupted prolactin signaling. These observations are consistent with the notion that elevated glycolysis and lactacidosis effectively disrupts prolactin-induced Nuc-pYStat5 in breast cancer.
The sensitivity of prolactin-induced signaling to acidosis is most likely due to a mechanism that involves protonation of histidine residues located at the ligand-receptor binding interface [
33,
49]. Four histidine residues are directly involved in high-affinity binding between prolactin and its cognate receptor based on crystal structures [
41]. In contrast, histidine residues are not critical for binding of the closely related but acidosis-resistant growth hormone to its cognate GHR [
50]. Mutational analyses have suggested that H180 of prolactin and H188 of PrlR are particularly important for the pH-dependent ligand-receptor binding. Importantly, human GH can also bind to hPrlRs and exert lactogenic activity [
51]. The binding of GH to hPrlRs is facilitated by a critical Zn
2+ binding site formed by two growth hormone residues (H18 and E174) and two prolactin receptor residues (D187 and H188) at the binding interface [
52]. Therefore, protonation of prolactin receptor H188 at acidic pH may interfere with Zn
2+-mediated binding of GH, and consequently disrupt the majority of GH-induced PrlR signaling in T47D cells. Only in the presence of supraphysiological concentrations of Zn
2+ (50 μM) did GH-induced signaling become resistant to acidosis in T47D cells. This effect is probably due to stabilization of the histidine imidazole group at high Zn
2+ concentration, which might protect PrlR H188 from becoming protonated. These observations are consistent with an earlier report based on a cell-free assay that the binding of GH to the PrlR extracellular domain was not pH-dependent in the presence high levels of Zn
2+ [
33]. Importantly, GH signaling through PrlRs, including proposed heterodimerization of PrlRs and GHRs [
53], is likely to remain pH
e-dependent at physiologic Zn
2+ levels. In contrast, GH activation of GHRs expected to be unaffected by acidosis.
In addition to the full-length or ‘long’ PrlR, alternative mRNA splicing generates ‘intermediate’ and ‘short’ PrlR isoforms that only differ in their cytoplasmic domain. Binding of prolactin to these PrlR isoforms is expected to remain sensitive to acidosis. On the other hand, binding of prolactin to the ΔS1 PrlR isoform may be less sensitive to acidosis due to its already poor ligand-binding kinetics caused by partial loss of the ligand-binding interface [
54]. Furthermore, 16 K prolactin is an N-terminal proteolytic fragment of prolactin that comprises only the first 145 amino acid residues. Therefore, 16 K prolactin lacks the critical H180 residue which mediates pH
e-sensitive binding of Prl to PrlR. Despite the well-documented anti-angiogenesis activity of 16 K prolactin, the receptor mediating its function remains to be identified, and the effect of acidosis on the function of 16 K prolactin is unknown. Interestingly, 16 K prolactin is generated by cathepsin D cleavage of full length prolactin [
55]. Cathepsin D is a lysosomal protease and thought to be active only at lysosomal pH range (approximately 5.0). More recent studies suggested that cathepsin D could be secreted and activated at acidic pH
e approximately 6.7 [
56]. Therefore, an acidic tumor environment might facilitate cleavage of full length prolactin into 16 K prolactin.
Acknowledgements
We thank Dr. Dennis Leeper and Mathew Thakur for helpful discussions and advice, and Jessica Davison for expert editorial assistance. This work was supported by Komen for the Cure Promise Grant KG091116 (HR, TH, JAH, AJK, CDS, TS, ARP, MAG, CL, BF and IC), NIH grants CA101841 and CA118740 (HR), and NCI Support Grant 1P30CA56036 to the Kimmel Cancer Center. The Project is funded, in part, under a Commonwealth University Research Enhancement Program grant with the Pennsylvania Department of Health (HR). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of the Army (DOA), Department of Defense (DOD), or US Government.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
NY, ARP, CL, TT, BF, IC, TH, CDS, JAH, AJK and HR conceived the study and participated in its design. JAH, AJK, CDS, THT and HR provided formalin-fixed, paraffin-embedded archived patient materials for the study. CL, MAG and ARP performed immunostaining, and quantitative immunofluorescence analyses. JAH, AJK and CDS conducted pathologic reviews and clinical data evaluations. ARP, BF, IC, TH, NY, ARP and HR performed statistical analyses. TT cultured three-dimensional T47D spheroids. FEU generated 32D-hPrlR and 32D-hGHR cell lines. AFY and NY conducted in vivo and in vitro experiments. NY and HR drafted the manuscript. All authors read, edited and approved the final manuscript.