As mentioned above, SSAs are a cornerstone medical treatment modality for NETs, targeting SSTR2 which is often expressed at a high level in NETs. The genomic DNA of human
SSTR2 contains multiple transcription start sites (TSSs). Two TSSs are located 82–93 nucleotides upstream [
51,
52] from the translation start codon with an initiation element
inr in close proximity. This
inr is involved in regulating gene transcription in the absence of a TATA-box as transcription factors are able to bind to the E-box present within this
inr [
53]. Another TSS is located further upstream and contains a CpG island [
54]. As CpGs are often the target for epigenetic modifications, it is likely that epigenetic regulation is involved in controlling
SSTR2 gene expression via this TSS. This suggests that deregulation of the epigenetic machinery may also influence tumoral SSTR2 expression. To elucidate the role of epigenetic regulation in NET patients, different NET cell lines have been used. These include cell lines derived from pNETs (i.e. BON-1 and QGP-1), pulmonary NETs (i.e. NCI-H727), siNETs (i.e. GOT-1) and medullary thyroid cancer (i.e. TT and MZ-CRC-1), which are all characterized by their own basal SSTR2 expression levels.
In pNET cells lines BON-1 and QGP-1, both DNA methylation and histone modifications regulate SSTR2 expression. In comparison with other NET cell lines, BON-1 and QGP-1 cells are both characterized by relatively low SSTR2 expression levels. However,
SSTR2 mRNA levels are still relatively high compared to cell lines derived from other types of cancer [
54]. QGP-1 cells demonstrated low
SSTR2 promotor methylation rates at only 2% in the 8 CpG islands examined [
55]. Similar observations were made for the pancreatic BON-1 cells, characterized by slightly higher SSTR2 expression levels compared to QGP-1 cells. Low (~3%), or even unmeasurable levels of DNA methylation were found in the genomic region surrounding the TSS in BON-1 cells [
54,
55]. The low levels of DNA methylation and relatively low SSTR2 expression levels could be related to the involvement of DNA methylation in other regions, as the above described studies only focus on specific areas in the promotor region. Torrisani et al. [
54] showed an inverse association between the level of CpG island methylation and
SSTR2 mRNA levels within several cell lines, including the pNET cell line BON-1. Additionally, transfection of a methylated
SSTR2 promotor in BON-1 cells induced silencing of the
SSTR2 promotor. This effect was caused by the absence of binding of transcription factor specificity protein-1, a protein involved in regulating the basal
SSTR2 promotor activity [
54]. Together, these observations support the potential of
SSTR2 promotor methylation to suppress SSTR2 expression. Moreover, acetylation on histone 3 was present in both BON-1 and QGP-1 cells [
56]. The involvement of histone acetylation was further confirmed by Veenstra et al. [
55]. In conclusion, both DNA methylation and histone acetylation are likely involved in regulating SSTR2 expression, i.e. triggering heterochromatin and euchromatin, respectively.
The above-mentioned associations between epigenetic markers and SSTR2 expression levels suggest that epigenetic drugs could potentially stimulate SSTR expression in NET cells. The use of epigenetic drugs, such as DNMTis and HDACis, may stimulate euchromatin, thereby promoting SSTR2 gene transcription. This approach can especially be important for NET patients not eligible for SSTR2-mediated therapies due to insufficient or undetectable SSTR expression levels.
3.1.1 Modulation of SSTR expression in vitro in NET cell lines
Successful stimulation of SSTR2 through attenuation of methylation has been demonstrated in BON-1 cells by treatment with DNMTis 5-aza-2′-deoxycytidine (5-AZA-dC) or 5-azacitidine (5-AZA), as shown by significantly enhanced uptake of [
68Ga]Ga-DOTA-TOC [
57]. Further analysis of 5-AZA-dC pretreatment demonstrated increased
SSTR2 mRNA and SSTR2 protein expression levels, which increased over time. Moreover, the uptake of [
68Ga]Ga-DOTA-TOC was clearly enhanced at human 5-AZA-dC therapeutic serum concentrations, whereas effects were barely observed at lower concentrations. Based on these data, a time- and dose-dependency was suggested. The efficacy of 5-AZA-dC in modulating SSTR2 expression is investigated in several other studies. A seven-day treatment schedule resulted in enhanced
SSTR2 mRNA expression levels in both BON-1 and QGP-1 cells using 100 nM and 50 nM, respectively. Receptor functionality was subsequently demonstrated with internalization studies using [
125I]I-[Tyr
3]octreotide, reporting a significantly increased 1.85-fold uptake in BON-1 cells [
55]. In line with this, significantly increased SSTR2 protein levels in BON-1 cells after a 3 day exposure to 2.5 μM 5-AZA-dC were also observed in the study by Jin et al. [
58]. However, in another study, it was demonstrated that a 3 day exposure to a lower dose of 5-AZA-dC (2 μM) had no significant effects on
SSTR2 mRNA expression levels in BON-1 cells [
54]. As the experimental set-up is similar in terms of cell line and exposure time, the results support data the above mentioned of Taelman et al. [
57] of a dose-dependent response. As both time- and dose-dependency are clearly suggested, a precise treatment regimen may be important parameter for study outcome.
In addition to DNA methylation, histone acetylation is also likely involved in regulating SSTR2. HDACis therefore also gained great interest as a novel therapeutic strategy to stimulate SSTR2 expression. Several HDACis have been tested in QGP-1 cells, which has led to contradictory results. Whereas Veenstra et al. [
55] demonstrated increased internalization of radiolabeled SSAs after valproic acid (VPA) treatment,
SSTR2 mRNA levels were significantly decreased by 1 mM VPA. This indicated other modes of action, e.g. fast redirection of the receptor to the membrane after internalization, via yet unknown epigenetic mechanisms. Contrary to these findings, significantly increased
SSTR2 mRNA levels were reported after VPA treatment by Guenter et al. [
59] when using a higher VPA dosage (4 mM). Other HDACis, such as romidepsin (FK228), vorinostat (SAHA) and AB3, provided similar results as significant upregulation was demonstrated on
SSTR2 mRNA expression level. Unfortunately, western blot analysis could not confirm SSTR2 upregulation in QGP-1 cells upon HDACi treatment [
59]. In contrast to this, the use of the HDACi LMK235 provided more convincing results, as this treatment resulted in increased SSTR2 protein expression levels [
56]. An epigenetic mechanism of action was confirmed by an augmented acetylation of histone 3 upon HDACi-treatment. Of note, LMK235 has high affinity for HDAC4 and HDAC5, both belonging to HDAC class IIa, whereas all the other tested HDACis either target multiple HDAC-classes or specifically target HDAC class I. This may suggest that HDAC4 and HDAC5 are highly involved in inducing euchromatin, thereby enabling
SSTR2 transcription in QGP-1 cells. The effects of HDACi-treatment in BON-1 cells were more consistent than the results in the QGP-1 cell line. A screen of several HDACis (i.e. scriptaid, dacinostat, panobinostat, trichostatin A (TSA), SAHA, phenylbutyrate, FK228 and tacedinaline (TAC)) demonstrated enhanced uptake of radiolabeled SSAs by BON-1 cells, reaching statistical significance for most HDACis [
57]. Further analysis of cells treated with TAC demonstrated significantly increased
SSTR2 mRNA and SSTR2 protein expression levels. In line with these results, significantly increased
SSTR2 mRNA levels were described after TSA treatment by Torrisani et al. [
54]. Furthermore, protein expression levels were significantly increased upon TAC treatment [
58], specifically inhibiting HDAC1–3, further supporting the enhanced uptake described by Taelman et al. [
57]. FK228, SAHA and AB3 were also able to enhance
SSTR2 mRNA significantly within 24 h, and even demonstrated increased protein expression levels after 48 h treatment [
59]. Moreover, it was shown that upon LMK235 treatment the level of acetylation on histone 3 was increased, providing a dose-dependent increase of SSTR2 protein after a one day treatment [
56].
Furthermore, the effects of VPA in BON-1 cells were evaluated in various studies. VPA treatment resulted in an increased level of acetylation on histone 4, thereby confirming changes in the epigenetic machinery. In line with this, SSTR2b protein expression was increased [
60]. This VPA-augmented
SSTR2 expression level was confirmed at mRNA level upon short- (24–28 h) and long-term (7 days) VPA treatment [
55,
59,
61]. Furthermore, a 7.2-fold stimulated SSTR2 protein expression level was observed [
59], while the functionality of increased SSTR2 expression was further confirmed by a significantly increased uptake of radiolabeled SSAs [
55,
57]. Receptor functionality was also confirmed by an increased efficacy of camptothecin-somatostatin conjugates after VPA treatment, as demonstrated by reduced BON-1 cell proliferation [
60]. These results suggest that SSTR2 expression is more easily modified in BON-1 cells than in QGP-1 cells, and the effects seem to be less dependent on the targeted HDAC classes.
In addition to pNETs, the effect of HDACis was examined in both small intestinal (i.e. GOT-1 and KRJ-I) and pulmonary (i.e. NCI-H727) NETs, characterized by variable SSTR2 expression levels. Expression levels in GOT-1 cells exceed that of NCI-H727 cells, which are both characterized by higher expression levels compared to BON-1 and KRJ-I cells [
58,
62]. Although there has been some debate about the origin of KRJ-I cells [
63,
64], VPA treatment increased
SSTR2 mRNA levels in these cells [
61]. Studies with the small intestinal cell line GOT-1 are still limited. The effect of HDACi treatment was solely examined upon monotherapy with either VPA [
61] or TAC [
58], resulting in a statistically significant ~2-fold increased SSTR2 protein expression level after TAC treatment, while no significant changes in mRNA expression were observed after VPA treatment. For comparison, a similar treatment schedule did change
SSTR2 significantly in BON-1 and KRJ-I cells.
In the pulmonary NET cell line NCI-H727, SSTR2 protein expression level was not changed significantly upon TAC treatment [
58]. However, in another study, HDACis thailandepsin-A (TDP-A), FK228, SAHA, VPA and AB3 were able to increase
SSTR2 mRNA levels significantly when high dosages were used [
65]. Western blot analysis confirmed over 2.5-fold upregulation in all conditions. Further examination of TDP-A-treated NCI-H727 cells demonstrated both receptor functionality and increased receptor-mediated uptake of [
68Ga]Ga-DOTA-TATE. Equal concentrations of TDP-A, FK228, SAHA, VPA, and AB3 showed SSTR2 protein upregulation in the TT medullary thyroid cancer cell line up to 3-fold, whereas these effects were not observed in the MZ-CRC-1 medullary thyroid cancer cell line. Of note, MZ-CRC-1 cells are characterized by high basal SSTR2 expression level compared to TT cells.
Studies have also focused on combining epigenetic treatments, e.g. the combination of DNMTis and HDACis. In BON-1 cells, the combination treatment of 5-AZA-dC and VPA had additive or synergetic effects as demonstrated by higher
SSTR2 mRNA levels and higher uptake of [
125I]I-[Tyr
3]octreotide compared to either monotherapy [
55]. Moreover, this combination of epigenetic drugs also significantly increased [
125I]I-[Tyr
3]octreotide uptake in QGP-1 cells, while effects of both monotherapies didn’t result in significant changes. In addition, combination of 5-AZA-dC and TAC gave synergistic effects as well, in terms of [
68Ga]Ga-DOTA-TOC uptake and cell survival [
57]. A similar combination treatment of 5-AZA-dC and TAC was examined in the BON-1, NCI-H727 and GOT-1 cell line by another research group, demonstrating significantly increased SSTR2 protein expression levels of 8.31-, 1.56- and 2.06-fold, respectively [
58]. Additive effects were demonstrated for BON-1 cells, whereas this was not evidently observed for H727 and GOT-1. Thus, the effects in H727 cells and GOT-1 cells were less pronounced compared to results obtained with BON-1 cells.
Altogether these studies clearly suggest the involvement of the epigenetic machinery in the regulation of SSTR2 expression in NET cells. Although convincing results in QGP-1 were only induced upon LMK235 treatment, results obtained with other cell lines suggest that especially NET cell lines with low (i.e. BON-1) or intermediate (i.e. NCI-H727 and TT cells) SSTR2 expression levels are susceptible to epigenetic drug treatment, whereas upregulation in NET cell lines with high SSTR2 expression levels is more limited (i.e. GOT-1 and MZ-CRC-1 cells). This supports the concept of epigenetic therapy for NET patients with insufficient SSTR2 expression, thereby potentially making more patients eligible for treatment with (radiolabeled) SSAs. The studies described above are summarized in Table
1. Of note, DNMTis (i.e. 5-AZA-dC) and some of the HDACis (i.e. VPA, TAC, SAHA, FK228) have been tested in clinical trials. Based on published pharmacokinetic parameters, it can be concluded that the drug concentrations used in the studies described above are within the same order of magnitude or even within the achievable human therapeutic range.
Table 1
Overview of in vitro studies with their main findings relevant for this review, focusing on modifying SSTR expression in NET cell lines using DNMTis or HDACis
BON-1 | Screen of several DNMTis and HDACis, e.g. 5-AZA-dC and TAC | 75 ng/mL 5-AZA-dC or 500 ng/mL TAC; time-dependency experiment (1–3 days) | - TAC and 5-AZA-dC increased the uptake of [68Ga]Ga-DOTA-TOC most efficiently; SSTR2 mRNA and SSTR2 protein expression levels were also significantly increased - Observed effects are time- and dose-dependent - Synergetic effects upon combination therapy in terms of [68Ga]Ga-DOTA-TOC uptake and cell survival | |
BON-1 QGP-1 | 5-AZA-dC, VPA | BON-1: 100 nM 5-AZA-dC and/or 2.5 mM VPA; 7 days QGP-1: 50 nM 5-AZA-dC and/or 1 mM VPA; 7 days | - Low SSTR2 CpG island methylation around transcription start site; ~3% in BON-1, ~2% in QGP-1 - All treatments increased SSTR2 mRNA levels and uptake of [125I]I-[Tyr3]octreotide significantly in BON-1; enhanced effects for combination therapy - SSTR2 mRNA levels and uptake of [125I]I-[Tyr3]octreotide increased after combination therapy in QGP-1 - Treatment of QGP-1 with VPA decreased SSTR2 mRNA levels and enhanced uptake of [125I]I-[Tyr3]octreotide non-significantly, suggesting other mechanisms of action - Histone acetylation more likely involved in regulating SSTR2 expression than histone methylation | |
BON-1 NCI-H727 QGP-1 GOT-1 | 5-AZA-dC, TAC | 2.5 μM or 5.0 μM 5-AZA-dC and/or 2.5 μM or 5.0 μM TAC; 3 days | - SSTR2 protein expression levels in QGP-1 undetectable before and after HDACi treatment - Combination treatment induced statistically significant upregulation of SSTR2 protein expression in BON-1, GOT-1 and NCI-H727; maximum increase of 8.31-fold in BON-1 - TAC significantly enhanced SSTR2 expression in BON-1 and GOT-1; 5-AZA-dC in BON-1 and NCI-H727 | |
BON-1 | 5-AZA-dC, TSA | 2 μM 5-AZA-dC and/or 150 nM TSA; 3 days | - SSTR2 upstream promotor not methylated - Significantly upregulated SSTR2 mRNA expression levels upon TSA and combination therapy - Statistically significant correlation between SSTR2 mRNA expression and CpG island methylation in upstream promotor | |
BON-1 QGP-1 | TDP-A, SAHA, VPA, FK228, AB3 | 2 nM or 6 nM TDP-1, 1 μM or 3 μM SAHA, 1 mM or 4 mM VPA, 2 nM or 6 nM FK228 or 1 μM or 3 μM AB-3 1 day for RT-qPCR, 2 days for further analysis | - SSTR2 mRNA levels significantly increased after 1 day treatment with 3 μM SAHA, 4 mM VPA, 6 nM FK228 and 3 μM AB3, in both BON-1 and QGP-1 - SSTR2 protein levels not evidently increased in QGP-1; maximum increase of 1.7-fold - SSTR2 protein levels clearly enhanced in BON-1; maximum increase of 7.2-fold - Increased functional SSTR2 density on cell surface for 6 nM FK228 in BON-1 | |
BON-1 QGP-1 | LMK235 | 0.08 μM, 0.31 μM, 1.25 μM, 5.0 μM and 20 μM; 1 or 2 days | - Dose-dependent increased acetylation on histone 3 upon LMK235 treatment - Dose-dependent increased SSTR2 protein level in BON-1 - SSTR2 protein levels detectable in QGP-1 after high concentration LMK235 treatment | |
BON-1 | VPA | 2 mM or 4 mM; time-dependency experiment (3, 6, 18, 36 and 72 h) | - Time-dependent increased level of acetylation on histone 4 - Reduced activity of HDAC4 after chronic treatment - Increased SSTR2b and decreased SSTR1, SSTR3, SSTR4 and SSTR5 protein expression levels - VPA enhanced anti-proliferating effects of camptothecin-somatostatin conjugates | |
BON-1 KRJ-I GOT-1 | VPA | 4 mM; 28 h | - Significantly increased SSTR2 mRNA expression level in BON-1 and KRJ-I | |
NCI-H727, MZ-CRC-1 TT | TDP-A, SAHA, VPA, FK228, AB3 | 2 nM or 6 nM TDP-1, 1 μM or 3 μM SAHA, 1 mM or 4 mM VPA, 2 nM or 6 nM FK228 or 1 μM or 3 μM AB-3 1 day for RT-qPCR, 2 days for further analysis | - SSTR2 mRNA levels significantly increased in NCI-H727 after highest-dose HDACi treatment; VPA and TDP-A also increased expression at lower dose - SSTR2 protein levels evidently increased; minimum increase of 2.5-fold in NCI-H727 - TDP-A treatment significantly increased uptake of [68Ga]Ga-DOTA-TATE in NCI-H727 - SSTR2 protein upregulated in TT after HDACi treatment; limited effects in MZ-CRC-1 which are characterized by higher basal SSTR2 expression levels compared to TT | |
3.1.2 Modulation of SSTR expression in vivo in NET xenograft-models
Based on the in vitro results discussed above, the effects of epigenetic drugs were also tested in vivo using NET-bearing mice. Direct anti-proliferative effects can be induced by HDACi treatment. Reduced xenograft growth was observed after AB3, VPA, TDP-A, FK229 and ENT treatment in BON-1, GOT-1, TT and/or H-STS NET tumor-bearing mice, although statistical significance was not reached in all studies [
49,
60,
61,
66‐
68]. The combination treatment of VPA and camptothecin-somatostatin conjugate significantly reduced BON-1 tumor growth by 66%, compared to 17% and 42% for both monotherapies, respectively [
60]. Tumors were not resected in this study and HDACi-upregulated SSTR2 expression was therefore not confirmed. Encapsulation of the HDACi TDP-A in micelles functionalized with either KE108 [
66] or octreotide [
68] reduced tumor volume with 92% and 74%, respectively. In both these studies, significant differences were found between the effects observed after treatment with TDP-A-loaded targeted micelles compared to TDP-A-loaded non-targeted micelles. Similar results were described for AB3-encapsulted KE108-functionalized micelles tested in medullary thyroid cancer TT xenografts [
67]. According to the authors, the enhanced effects for TDP-A-loaded targeted micelles can be explained by the combination of both passive and active tumor targeting ability, i.e. enhanced permeation retention effect and efficient targeting of SSTRs, respectively. Unfortunately, tumors were not further analyzed to confirm changes in SSTR2 expression levels upon HDACi treatment. Although these data are not available, it may also be hypothesized that the enhanced effects upon treatment with TDP-A-loaded or AB3-loaded targeted micelles are caused by the fact that the HDACis are targeted to the SSTR2-expressing tumor cells, resulting in enhanced receptor expression due to HDACi-mediated changes in the epigenetic machinery. This SSTR2 upregulation may lead to increased therapeutic efficacy as more functionalized micelles will be targeted to the tumor cells. However, this hypothesis requires further investigations.
In the study published by Taelman et al. [
57], it was demonstrated that 5-AZA-dC significantly increased the uptake of [
68Ga]Ga-DOTA-TOC in BON-1 tumor-bearing mice in a dose-dependent manner, resulting in increased tumor-to-background and tumor-to-kidney ratios. Moreover, a blocking study demonstrated SSTR-specific uptake after HDACi treatment, indicating SSTR-upregulation. As a result, tumors could be visualized using PET/CT-imaging modality. In addition to this study using a DNMTi, two studies have been published in which SSTR2 expression levels were examined by PET/CT-scans upon inhibition of HDAC class I proteins. For BON-1 tumor-bearing mice, significantly increased standard uptake values (SUVs) were observed on a PET/CT-scan after [
68Ga]Ga-DOTA-TATE injection when mice were pre-treated with FK228 [
59]. A similar effect was observed in mice with NCI-H727 xenografts that were treated with TDP-A. This study showed a trend towards SSTR upregulation following HDACi-treatment, although statistical significance was not reached due to differences in individual tumor size and uptake of [
68Ga]Ga-DOTA-TATE [
65].