NIS expression in breast cancer
The demonstration of NIS presence in lactating breast [
23] has suggested that this protein could be expressed also in breast cancer (BC). Accordingly, in the seminal study in which NIS expression in lactating breast was discovered, it was shown that this protein is expressed in more than 80% of both invasive and
in situ BCs [
23]. However, both plasma membrane and intracellular immunohistochemical signal was detected (Table
2), which is in contrast to the only basolateral membrane signal detected in lactating breast. The notion that the NIS protein is expressed in a large number of breast carcinomas was confirmed by the same group by investigating a larger cohort of samples [
13]. In this study it was found that NIS is also expressed in about 80% of fibroadenomas. Again, in breast carcinomas, the NIS protein was predominantly located in the cytoplasm, suggesting that in BC a deficiency of NIS trafficking from cytoplasm to plasma membrane occurs. High levels of NIS positivity in BC by immunostaining has also been described in other studies [
31,
32]. It should be pointed out, however, that such a large positivity, when obtained by immunohistochemistry in the cytoplasmic compartment, could be due to non-specific staining [
33]. In order to understand the molecular basis of NIS inability to target the plasma membrane in a large fraction of BC, genes whose expression is associated to NIS plasma membrane localization have been recently identified by microarray analysis [
34]. Interestingly, the cysteinyl-tRNA synthetase gene is highly associated with cell surface NIS protein levels only in the estrogen receptor (ER)-positive BC subtype, suggesting that molecular mechanisms responsible for reduced plasma membrane localization of NIS may be different in a distinct subtype of BC [
34].
Table 2
NIS expression in breast cancer tissues
45 | n.d. | 69 | |
50 | n.d. | 90 | |
12 | + | 100 | |
27 | n.d. | 30 | |
23 | n.d. | 65 | |
28 | + | 7 | |
75 | + | n.d. | |
32 | n.d. | 92 | |
Triple-negative BCs (TNBCs) are defined by the absence of ER, the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2) expression [
38]. Because of absence of ER, PR, and HER2, TNBC cannot be treated by hormonal therapy or HER2-targeting compounds, leaving chemotherapy as the only therapeutic tool. Patients with this disease have a worse outcome than patients with other BC subtypes [
38,
39]. It has been shown that NIS is expressed in about 65% of TNBCs and that in a fraction of them a strong plasma membrane localization is present [
40]. Accordingly, in the same study, efficient iodine uptake was detected by
123I scintigraphy in a patient. The notion that the NIS protein expressed in BC is able to allow radioiodine uptake has been reported in other studies as well. In fact, by studying women with infiltrating duct carcinoma, high NIS expression at both transcriptional and translational level and its ability to transport iodine in cancer tissue has been demonstrated [
35]. Recently, Damle and coworkers reported that the radioiodine uptake in breast cancer specimens was significantly higher as compared to that observed in the normal tissue from the same patients [
41]. In this study, 50% of breast cancer samples were positive for radioiodine uptake as well as
NIS gene expression [
41].
Expression and function of NIS has been investigated also in metastatic BC. Wapnir and coworkers investigated 23 patients with metastasis predominantly at the level of lung, liver, bone, and lymph node/soft tissues [
30]. Eight of these subjects showed protein NIS expression, and iodide uptake was noted in two of eight NIS-expressing tumors. The same group has more recently investigated NIS expression in brain metastasis by immunohistochemistry [
40]. In 75% of cases a predominant cytoplasmic signal was detected; however, plasma membrane immunoreactivity was detected only in 23.8% of NIS-positive samples. Altogether these data would indicate that NIS protein is correctly located and is able to accumulate iodine only in a small fraction of BC metastasis.
Besides immunohistochemical studies, high expression of NIS mRNA has been shown by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) evaluation. Oh and coworkers have shown that
NIS gene expression was present in approximately one-third of BC tissues, and no relationship was found between NIS mRNA levels and hormonal receptors expression [
42]. More recently, Ryan et al. confirmed that NIS expression levels are significantly higher in BC and fibroadenoma than in normal tissue, with the highest levels of NIS mRNA observed in fibroadenoma tissues [
37]. At present, detection of NIS expression levels has no prognostic value: in fact no significant relationship has been detected between NIS mRNA levels and clinical characteristics of the tumors [
37]. In addition, immunohistochemistry of a subset of tumor tissues in the same cohort confirmed the presence of NIS protein both in selected malignant carcinomas and benign fibroadenomas [
37].
NIS-based gene therapy
A strategy attempted to achieve significant radioiodine uptake by the BC cells is using gene therapy to introduce an "active" exogenous
NIS gene. Montiel-Equihua and coworkers have generated a replication-incompetent adenovirus, AdSERE, in which the expression of NIS is directed by an estrogen-responsive promoter [
43]. Therefore, this vector would be active only in ER-positive BC (about 60% of all BC).
In vitro, AdSERE mediated human NIS expression and iodide uptake in ER+ cell lines (MCF7 and ZR75-1). Moreover, the authors show that ZR75-1 AdSERE-positive xenografts in nude mice can be imaged after
99mTc injection and their growth suppressed with therapeutic doses of
131I [
43]. The use of a non-replicative adenovirus has been recently reported by the Santisteban group [
44]. In this virus, NIS transcription is driven by promoters of human telomerase subunits RNA (hTR) and human telomerase reverse transcriptase (hTERT). Telomerase is a ribonucleoprotein that is essential in most human cancers but is not expressed in most normal tissues [
45‐
47]. Thus, hTR and hTERT promoters would be active only in cancer cells. When the BC cell line MDA-MB-231 was infected by this virus, expression of NIS protein, iodine uptake, as well as reduced cell survival after radioiodine administration was observed. A conditionally replicating adenovirus (CRAd) in which the E1a gene is driven by the tumor-specific promoter Mucin 1 (MUC-1) has also been generated [
48]. This virus can efficiently replicate only in MUC-1 overexpressing cells, including BC cells [
49]. In addition, this virus contains the transcriptional cassette RSV promoter-h
NIScDNAbGH polyA in the E3 region, which permits NIS to express at high levels. After infection of the MUC-1-positive BC cell line T47D, virus replication, cytolysis, and release of infective viral particles, as well as iodide uptake, were observed [
48].
The increase of the exogenous, virus-mediated expression of the
NIS gene by pharmacological treatment has been also investigated. Treatment with retinoic acid (RA) has been shown to increase NIS expression in MCF7 cells infected by a non-replicating adenovirus in which NIS expression is controlled by the potent cytomegalovirus (CMV) promoter [
50]. Indeed, the CMV promoter contains an RA-responsive element [
51]. A large increase of iodine uptake has been also described in virus-infected, RA-treated MCF7 cells.
Induction of endogenous NIS
Though NIS expression has been demonstrated in most BCs, only in very few patients would spontaneous NIS expression allow efficient radioiodine uptake. For this reason, a large body of investigation has been undertaken to identify compounds that are able to increase NIS expression, its localization in plasma membrane, and iodine uptake. The major inductor of NIS expression in breast cancer cells is certainly RA. Several compounds of the RA family stimulate NIS expression, including all-
trans RA, 13-
cis RA, and AGN190168, all of which are already used for medical purposes [
9]. Among them, the one used most to activate NIS expression in BC cells is all-
trans RA. NIS expression has been induced in several BC cell lines including MCF7, T47D, and BT474 [
52]. Several data indicate that RA induces NIS expression primarily by activating RARβ/RXRα heterodimer receptors. Hormone-bound receptor may act through two mechanisms. The first is binding to an element located in
cis to the
NIS gene [
9]. It has been demonstrated that in MCF7 cells, treatment by RA induce retinoic acid receptor-alpha (RARα) binding to a retinoic acid response element located in intron 2 of the
NIS gene [
53]. It must be mentioned, however, that the NIS regulation by RARα was not confirmed in a different study performed on MCF7 cells [
9]. The second mechanism is activation of the phosphoinositide 3-kinase (PI3K) pathway and the p38MAPK pathway. In MCF7 cells, Ohashi and coworkers have shown that either treating cells with the PI3K inhibitor LY294002 or inducing knockdown of p85alpha (a regulatory subunit of PI3K) decreases RA-induced NIS expression. Moreover, the AKT inhibitor VIII decreases iodine uptake in MCF7 cells in a dose-dependent manner [
54]. Kogai and coworkers, by using both gain and loss of function experiments, have shown that p38β plays a role in the RA-induced NIS expression increase in MCF7 cells [
55]. Interestingly, in the same study it was shown that in FRTL5 thyroid cells not the β but the p38α isoform has a role in NIS control of expression. Moreover, NIS induction was also observed in mouse MCF7 xenograft [
56,
57], although this finding was not confirmed by another group [
58]. These different results using MCF7 cells might be due to heterogeneity of this cell line [
59].
In addition to gene expression, the PI3K pathway regulates NIS localization. Glycosylation of NIS protein is necessary to plasma membrane localization [
60]. In MCF7 cells, overexpression of PI3K increases the non-glycosylated NIS protein [
61]. In the same study, it was shown that the presence of NIS in the plasma membrane as well as iodine uptake was reduced by an active mutant of PI3K. It appears, therefore, that activation of the PI3K signaling pathways exerts opposite effects on NIS: expression is activated while NIS localization in the plasma membrane is inhibited.
Several compounds cooperate with RA in inducing NIS expression in BC cell lines. RA-induced enhancement of NIS is increased by hydrocortisone, dexamethasone, troglitazone (a peroxisome proliferator–activated receptor γ, PPARγ, agonist), histone deacetylase (HDAC) inhibitors (tricostatin A and sodium butyrate), and carbamazepine [
58,
62‐
64]. Hydrocortisone, dexamethasone, troglitazone, and carbamazepine cooperate with RA also in inducing iodine uptake. Interestingly, by using MCF7 xenografts in nude mice, it has been shown that RA alone is not able to increase iodine uptake; however, significant increase in
123I accumulation occurs when RA is used in combination with dexamethasone [
65]. Other stimulators, such as prolactin, insulin, and insulin growth factor (IGF)-I and II, are able to increase NIS mRNA levels in MCF7 cells also in the absence of RA [
66]. Fortunati and coworkers reported that the HDAC inhibitor LBH589 significantly induced NIS mRNA and protein levels as well as iodine uptake in several BC cell lines [
67]. Table
3 summarizes the data regarding the stimulation of iodide uptake in breast cancer cells.
Table 3
Stimulators of iodide uptake in breast cancer cell lines
MCF7 | tRA,9-cis RA | RAR/RXR agonist | 10 ~ 13 | |
MCF7 | AGN190168 | RARβ/γ agonist | 10 ~ 13 | |
MCF7 | Am80 | RARα/β agonist | ~7 | |
MCF7 | Theophylline | PDE antagonist/P2R inhibitor | ~4.7 | |
MCF7 | LBH589 | HDAC inhibitor | ~2.3 | |
T47D | LBH589 | HDAC inhibitor | ~4.8 | |
MDA-MB231 | LBH589 | HDAC inhibitor | ~2.7 | |
MCF7 | Insulin | Insulin receptor | ~12 | |
MCF7 | IGF-I | IGF-I receptor | ~7.8 | |
MCF7 | IGF-II | IGF-II receptor | ~10.3 | |
MCF7 | Prolactin | Cytosolic PKs activation | ~9 | |
MCF7 | Forskolin | Adenilyl-cyclase/PKA activation | ~3.1 | |
MCF7 | TPA | PKC activation | ~2.6 | |
MCF7 | (Bu)2-cAMP | PKA activation | ~3.4 | |