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DOI: 10.1530/eje.1.02077
European Journal of Endocrinology, Vol 154, Issue 1, 159-166
Copyright © 2006 by European Society of Endocrinology
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EXPERIMENTAL STUDY

RT-PCR analysis of corticotroph-associated genes expression in carcinoid tumours in the ectopic-ACTH syndrome

M Messager, C Carrière, X Bertagna and Y de Keyzer1

Institut Cochin, INSERM U567-CNRS UMR8107, 24 rue du faubourg Saint Jacques, 75014, Paris, France and 1 INSERM U393, Hopital Necker, 149 rue de Sèvres, 75015 Paris, France

(Correspondence should be addressed to Y de Keyzer; Email: dekeyzer{at}necker.fr)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: ACTH is frequently produced in non-pituitary tumours, leading to the ectopic-ACTH syndrome, but the molecular mechanisms of its expression remain obscure. This study was aimed at understanding the transcription mechanisms of the ACTH-precursor gene in carcinoid tumours of the lung or thymus.

Design: Transcripts coding for a series of corticotroph-associated transcription factor genes were detected, together with markers of the corticotroph phenotype. We studied a series of 41 carcinoid tumours including 15 with proven ectopic-ACTH syndrome.

Methods: Specific RT-PCR reactions were designed for each gene including alternatively spliced isoforms.

Results: The markers of the corticotroph phenotype were detected in all ACTH-positive tumours. Expression of the Tpit and Pitx1 genes were not restricted to ACTH-positive tumours but were also detected in many ACTH-negative carcinoids. Only a subset of ACTH-negative tumours expressed NAK-1/Nur77, and NeuroD1 expression was detected in <50% of the tumours regardless of their secretory status. The glucocorticoid receptor alpha was detected in every tumour in contrast to its beta isoform detectable in a few tumours only. Chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and peroxisome proliferator-activated receptor (PPAR) {gamma}2 were expressed in 50% of the tumours of each group whereas PPAR{gamma}1 was expressed in almost every tumour.

Conclusions: ACTH-positive carcinoids do not share a characteristic expression pattern of the corticotroph-associated transcription factor genes, suggesting that the transcriptional mechanisms of the ACTH-precursor gene differ from those in normal pituitary corticotrophs. Expression of Tpit and Pitx1 genes in most carcinoids suggests that some aspects of the pituitary corticotroph phenotype may belong to general carcinoid differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Proliferating tumour cells often maintain differentiated functions that are part of their normal counterparts. In this view, endocrine features are usually found in tumours deriving from endocrine glands or tissues known to contain neuroendocrine cell populations (1, 2). Tumoral hormone production is frequently altered leading to inappropriate secretion of biologically active hormone and, eventually, to clinical manifestations which worsen the patient’s condition. Occasionally, tumours developed from tissues considered as non endocrine may express a given hormone gene and some even secrete large amounts of biologically active hormone, eventually leading to ectopic hormone secretion syndromes. Whereas any hormone gene may theoretically be expressed in tumours, only a limited subset of hormones are actually secreted to levels high enough to induce clinical manifestations, and adrenocorticotrophin (ACTH) is one of the most frequently reported. ACTH production by human non-pituitary tumours was recognized a long time ago (3) and is now known as the origin of the ectopic-ACTH syndrome (EAS); however, the molecular bases for this ectopic production remain unknown. Proopiomelanocortin (POMC, the precursor to ACTH) gene expression can be detected at the mRNA level in many non-pituitary tissues, either tumoral or not, but as a truncated ca. 800 nt transcript limited to exon 3 sequences, thus lacking the 5' extremity of the coding region and unable to translate into a functional polypeptidic precursor molecule (4–6). In contrast, in tumours associated with EAS, POMC mRNA contains the full coding region and is detected as a pituitary-like 1200 nt transcript. We have previously shown that the structure of this RNA is identical to that of POMC mRNA in the pituitary (7, 8), suggesting that the same promoter region is used for transcription in these non-pituitary tumours and normal pituitary corticotrophs. Furthermore, studies in various ACTH-secreting tumours have shown that the POMC promoter region is unaltered (9). It is of clinical and fundamental interest to unravel the molecular mechanisms of POMC gene transcription in non-pituitary tumours, in order to understand how a tumoral cell acquires the ability to produce ACTH, and why the physiological regulators of ACTH secretion and POMC gene expression are usually not efficient in these cells.

The differentiation of the corticotroph lineage in the pituitary has been studied for many years and a series of transcription factors has been identified that participate both in the differentiation and in POMC gene expression in mature cells. Among the many factors that bind the POMC promoter, tissue-specific expression involves Pitx1, NeuroD1, Nur77 and Tpit (10–14), and the transcriptional negative regulation is principally mediated by the glucocorticoid receptor (GR). In addition, recent studies on corticotroph pituitary tumours also identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and peroxisome proliferator-activated receptor gamma (PPAR{gamma}) as important regulators of both POMC gene expression and tumour cell proliferation (15, 16). Although these factors are not specific to corticotroph tumours (17), their identification opens new perspectives to understand the molecular mechanisms involved in POMC gene expression and regulation in human tumours.

Two classes of non-pituitary tumours can be distinguished with respect to POMC gene expression characteristics: (i) highly proliferative tumours, such as small cell lung carcinomas (SCLC), where the 1200 nt POMC mRNA is present at low levels with frequent qualitative alterations and where POMC expression is not accompanied by other pituitary corticotroph specific markers such as the type-3 vasopressin (V3R or V1bR) receptor; (ii) well differentiated tumours such as carcinoids, which often display high levels of the 1200 nt POMC mRNA together with high levels of the V3R receptor mRNA (18, 19).

In order to provide insights into the mechanisms of non-pituitary tumour expression of the POMC gene, we analysed the expression of several corticotroph-associated transcription factors in a large series of carcinoid tumours originating mostly from bronchial epithelium together with corticotroph markers used to characterize their differentiation. We also examined the mRNA level of several transcription factors involved in abnormal POMC gene regulation in corticotroph pituitary tumours, such as PPAR{gamma} and COUP-TF1.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Tissue collection

Tumour samples were collected during surgery at the Centre chirurgical Marie Lannelongue (Le Plessis-Robinson, France), with informed consent of the patients. The patients were not under octreotide treatment at the time of surgery. The tissue was immediately frozen in liquid nitrogen and stored at –80 °C. A series of 41 tumours was selected including 15 responsible for EAS as determined by blood cortisol and ACTH concentration measurements after classical dynamic tests. The presence of ACTH in the tumours was established by immunohistochemistry with a monoclonal antibody directed against the 24–39 part of ACTH (clone 02A3, DAKO Corp., Carpinteria, CA, USA) by pathologists. In the few cases where such data were not available, ACTH was directly assessed in tumour extracts with a radioimmunoassay developed in our laboratory (20) and RNA analysis in our laboratory. Tumours were classified as atypical or typical after histological examination by the pathologists, and as centrally or peripherally located by the surgeon (Table 1Go). Normal pituitaries were obtained during autopsy between 12 and 24 h post mortem and stored at –80 °C.


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  		Table 1 Clinical data of the patients and tumors studied.
 
RNA extraction

Total RNA was extracted by the guanidium/cesium chloride method as described (21) and treated with 1 unit DNase I for 30 min at 37 °C to remove contaminant genomic DNA traces. RNA concentrations were determined by absorbance at 260 nm and RNA integrity was checked by electrophoresis.

RT-PCR analysis

RNAs (2 µg) were reverse transcribed for 1 h at 42 °C with 200 U superscript II transcriptase (Invitrogen) and 25 ng/µl poly-dT(12–18). RT-PCR oligodeoxynucleotides were designed for the human sequences of POMC, V3R, the type-1 corticotrophin releasing factor receptor (CRHR1), the membrane proteins KIAA1775 and TM4SF5 (22), glyceraldehyde-3-phosphate de-hydrogenase (G3PDH), and the transcription factors Pitx1, neuroD1, Tpit, NAK-1 (the human orthologue of Nur77, hereafter called NAK-1/Nur77), GR{alpha}, GRß, COUP-TF1, PPAR{gamma}1 and PPAR{gamma}2 (Table 2Go). They were used to amplify the cDNA preparations by PCR for 23–35 cycles composed of 30 s at 94 °C, 30 s annealing and 30–60 s at 72 °C (Table 2Go) followed by a final 5 min incubation at 72 °C in a 50 µl reaction. All primer pairs were selected to encompass at least one intron in order to easily distinguish the products amplified from genomic DNA from those amplified from cDNA templates.


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  		Table 2 Characteristics of the PCR reactions.
 
Each PCR was performed with 10% of the cDNA reaction and the PCR products (10 µl) were analysed on 1–2% agarose gels containing ethidium bromide. The nucleotide sequences of the various PCR products were established by the dideoxy-nucleotide chain termination method to ascertain the identity of the amplified product.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Analysis of corticotroph and carcinoid markers

Two groups of carcinoid tumours, mostly from bronchial origin, were constituted on the basis of Cushing’s syndrome diagnosis. The clinical features of the 15 ACTH-secreting (ACTH-positive) tumours, including two thymic carcinoids, and the 26 non-ACTH-secreting (ACTH-negative) tumours are reported in Table 1Go. We first characterized these tumours with respect to their corticotroph phenotype (Fig. 1Go). As expected, a strong RT-PCR signal was detected for POMC in all tumours responsible for EAS, whereas it was not or was barely detectable in the ACTH-negative group. Pituitary samples (left panel) used as controls yielded a signal in both normal (left lane) and corticotroph tumoral tissue (right lane), with the expected higher signal in the tumour sample. A strong signal for V3R was detected in all but one of the ACTH-positive carcinoids as compared with the very weak bands observed in the ACTH-negative tumours. Expression of CRHR1, another marker of corticotroph differentiation in the pituitary, was observed in most, but not all, ACTH-positive carcinoids and was also detected, sometimes as an intense signal, in about half of the ACTH-negative group. Both V3R and CRHR1 genes were expressed in pituitary tissues, with a stronger signal in the corticotroph tumour as already reported (21, 23, 24). We took advantage of the recent characterization of KIAA1775 and TM4SF5, two genes specifically or preferentially expressed in ACTH-positive or ACTH-negative carcinoids respectively, to further substantiate the phenotype of both groups. KIAA1775 expression was only detectable in ACTH-positive carcinoids, although not in every one, and the signal intensity was highly variable. The RT-PCR signal was much more intense in the pituitary corticotroph tumour sample compared with the normal pituitary tissue. On the other hand, TM4SF5 expression was detectable as a strong signal in most ACTH-negative tumours and only yielded a weak signal in four ACTH-positive carcinoids. A very faint signal was observed in the pituitary corticotroph tumour, whereas TM4SF5 expression could not be found in the normal pituitary. On these grounds, both carcinoid groups appeared homogeneous and displayed the expected characteristics related to their secretory status.



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  		Figure 1 RT-PCR analysis of the expression of corticotroph and carcinoid markers. The genes are indicated on the left. Tumour groups are indicated at the top (ACTH-positive, ACTH+; ACTH-negative, ACTH–). *Thymic carcinoids; Pit, pituitary samples: left lane normal human pituitary, right lane corticotroph pituitary tumour.

 
Analysis of corticotroph-associated transcription factors

We analysed the expression of four transcription factors involved in basal corticotroph-specific POMC gene transcription in the pituitary (Fig. 2Go).



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  		Figure 2 RT-PCR analysis of the expression of corticotroph-associated transcription factors in carcinoid tumours. The genes are indicated on the left. Tumour groups are indicated at the top (ACTH-positive, ACTH+; ACTH-negative, ACTH–). *Thymic carcinoids; Pit, pituitary samples: left lane normal human pituitary, right lane corticotroph pituitary tumour.

 
The homeobox factor Pitx1 was detected as a strong signal in most tumours, regardless of their corticotroph phenotype. It should be noted, however, that the two ACTH-positive carcinoids from thymic origin did not express detectable levels of Pitx1 mRNA, whereas only one ACTH-positive bronchial tumour displayed a weak Pitx1 signal. As expected for this pan-pituitary factor, its expression was detected in both normal and tumoral pituitary samples.

The expression of the NeuroD1 gene was detected in approximately 50% of the tumours, without preferential association with one group. The signal intensity was highly variable and strong signals were only observed in 4 ACTH-positive and 5 ACTH-negative carcinoids. Pituitary samples showed the expected signal, with an apparent increase in the corticotroph tumour.

The T-box binding factor, Tpit, was expressed in almost every sample of both tumour groups. In contrast, NAK-1/Nur77 expression was only barely detectable in two ACTH-positive bronchial carcinoids and detectable in about 1/3 of the ACTH-negative samples.

Analysis of transcriptional regulators

In the pituitary, glucocorticoids are the major negative regulator of both POMC gene transcription and ACTH secretion and their effects are, at least in part, mediated by the glucocorticoid receptor (GR). Two GR forms have been identified, the classical and active GR{alpha} and its splice variant GRß, which displays a dominant negative activity. We examined the expression of both GR mRNA species (Fig. 3Go). The expression of GR{alpha} mRNA was detected in all carcinoid samples. On the other hand, GRß expression was undetectable in all tumours except for one ACTH-negative tumour where a weak signal was detected. COUP-TF1 expression was observed in approximately 50% of the tumours, regardless of their secretory status, with a similar proportion in both groups (Fig. 3Go). In agreement with a previous report, COUP-TF1 mRNA was present in the normal pituitary and absent in the pituitary tumour used as control (15).



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  		Figure 3 RT-PCR analysis of the expression of POMC-gene transcriptional regulators in carcinoid tumours. The genes are indicated on the left. Tumour groups are indicated at the top (ACTH-positive, ACTH+; ACTH-negative, ACTH–). *Thymic carcinoids; Pit, pituitary samples: left lane normal human pituitary, right lane corticotroph pituitary tumour.

 
Two PPAR{gamma} isoforms, PPAR{gamma}1 and PPAR{gamma}2, have been identified (25) resulting from differential promoter and alternative splicing combinations. Both isoforms were expressed in our tumour series; PPAR{gamma}1 RT-PCR product was almost constantly detected whereas PPAR{gamma}2 was detectable, generally as a weaker signal, in about half of the samples regardless of their ACTH status.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
ACTH secretion is the hallmark of the corticotroph differentiation; it results primarily from POMC gene expression in specialized endocrine cells and is secondarily dependent on the presence of a subset of proteolytic enzymes able to cleave the POMC precursor molecule into ACTH. Studies in the murine corticotroph cell line, AtT20, have identified a series of transcription factors involved in basal corticotroph-specific POMC gene transcription and a general although still incomplete mechanism is emerging.

The expression of the transcription factors acting on POMC gene promoter is maintained in pituitary corticotroph tumours, suggesting that the overall mechanism of POMC gene transcription is also conserved. However, why and how POMC gene is expressed in ACTH-producing non-pituitary tumours remains obscure. Studies on highly proliferating neuroendocrine tumour models such as SCLC cell lines suggested that some pituitary corticotroph factors are not present and that POMC transcription is achieved through aberrant mechanisms, involving factors activated during tumoral transformation such as E2F (26, 27). This result was also consistent with the common observation that such poorly differentiated tumours lack most of the corticotroph markers and only express the POMC gene at low or very low levels.

However, due to the lack of in vitro models, similar studies have not been possible with slowly proliferating and well differentiated carcinoid tumours, yet they represent the most common origin of EAS. The results reported here represent the first attempt to compare the expression of a series of genes related to the corticotroph phenotype in ACTH-positive and ACTH-negative carcinoids. We confirmed and extended previous observations showing that the corticotroph phenotype of ACTH-positive carcinoid tumours is not limited to POMC gene expression but also includes cell surface receptors such as the V3R. The low level of V3R expression detected in ACTH-negative carcinoids most likely corresponds to the low level of expression already reported for all three vasopressin receptor subtypes in most human tumours (28), and thus should not be considered as an indicator of corticotroph differentiation. Similarly, CRHR1 expression had already been shown not to be strictly associated with the corticotroph phenotype and our results further establish this on a large series of tumours (21, 29). In addition, the pattern of CRHR1 expression in ACTH-negative carcinoids may suggest either that some tumours have a partial corticotroph differentiation or that CRHR1 expression is a common feature of neuroendocrine tumours or tumours in general. No association could be established between the subset of CRHR1-positive carcinoids and a particular combination of corticotroph-associated transcription factors, either in ACTH-positive or in ACTH-negative tumours.

We also analysed KIAA1775 and TM4SF5 gene expression since these genes were identified in carcinoid tumours through their association with the ACTH-positive and ACTH-negative phenotypes respectively (22). It is worth noting that TM4SF5 is, so far, the only gene that allows the characterisation of the ACTH-negative carcinoids by a positive criterion, and not just through the absence of the corticotroph markers analysed. Again, the results confirmed our previous observation, but similarly to CRHR1, their expression could not be linked to a given combination of corticotroph-associated transcription factors.

Of the four corticotroph-associated transcription factors examined only Tpit expression has been shown, so far, to be restricted to pituitary corticotrophs and this holds true for pituitary tumours (30). Surprisingly, this specificity was not conserved in carcinoids where it was expressed in ACTH-positive as well as in ACTH-negative carcinoids, suggesting that other essential factors are required to achieve a complete corticotroph differentiation. Furthermore, to our knowledge this is the first report of Tpit expression outside of the pituitary but also in non-corticotroph cells (11, 28). It has been shown that in the context of pituitary cells, also expressing Pitx1, expression of a Tpit transgene is able to induce POMC gene transcription (14), yet the simultaneous transcription of Pitx1 and Tpit in carcinoid tumours was not sufficient to do so. The co-expression of both factors in most bronchial carcinoids may, however, provide a transcriptional background favouring the expression of POMC and other corticotroph markers if additional corticotroph-associated factors become expressed. Similarly to Pitx1, NAK-1/Nur77 and NeuroD1 are unlikely to play this key role because only some ACTH-negative tumours and none of the ACTH-positive tumours expressed all four factors, indicating that this combination is not sufficient per se to induce the transcription of pituitary-like POMC mRNA. Alternatively, it is also possible that different mechanisms relying on unidentified factors are used in these tumours to differentiate towards the corticotroph phenotype or that inhibiting factors block the activity of Pitx1 and Tpit on the POMC promoter in ACTH-negative tumours. Our results also suggest that NeuroD1 and NAK-1/Nur77 are not instrumental for corticotroph differentiation and are likely to be replaced by other factors in ACTH-positive carcinoids. However, the frequent absence of NAK-1/Nur77 in ACTH-positive tumours was unexpected since it plays important roles in POMC gene transcription and is also involved in the negative regulation of POMC gene expression by glucocorticoids, which is one of the major clinical concerns in EAS (12, 31–33). The detection of GR{alpha} mRNA in every tumour indicated that GR{alpha} is not sufficient for the negative regulation of POMC promoter and suggested that additonal factors, needed for the negative regulation of POMC gene transcription, are missing in ACTH-positive tumours. GRß, a naturally occurring dominant negative isoform of GR{alpha} was not detected in ACTH-positive tumours, indicating that it is not involved in the resistance to glucocorticoid feedback. Although we did not look for GR{alpha} mutations, it is unlikely that mutations are a common cause for the lack of glucocorticoid sensitivity in ACTH-positive carcinoids since inactivating mutations have only been exceptionally reported in ACTH-secreting tumours of any origin (34–36).

Recent studies reported the inhibitory effects of retinoic acid (RA) and thiazolidinediones (TZD) on POMC gene expression and cell proliferation in pituitary corticotroph tumour cells (15, 16). The inhibition by RA correlated with the loss of COUP-TF1 expression in the tumours whereas the sensitivity of tumour cells to TZD correlated with the appearance of PPAR{gamma} in tumour cells only. Few tumours actually expressed the COUP-TF1 gene, which only represents the minimal requirement for RA action, suggesting that RA is unlikely to affect POMC gene transcription in most ACTH-positive carcinoids. Conversely, the expression of both PPAR{gamma} isoforms in almost every tumour suggests that TZD may exert some action on POMC expression. Finally, the two thymic carcinoids included in the ACTH-positive group could not be distinguished from bronchial ones except for Pitx1 expression, suggesting that a comparable corticotroph differentiation is reached in these two tumours. However, this observation needs to be confirmed on a larger series of thymic carcinoids, including ACTH-negative ones.

In conclusion, POMC gene expression in ACTH-positive tumours was not associated with a characteristic expression pattern of corticotroph-associated transcription factor genes. The expression of Tpit and Pitx1 genes in almost all carcinoids suggests that some aspects of the normal pituitary corticotroph phenotype may be part of general carcinoid differentiation, a possible explanation for the high prevalence of carcinoids among the tumours responsible for EAS. Despite their absence of specificity, TZD or other PPAR gamma ligands may represent new directions in the management of ACTH-positive carcinoids.


   Acknowledgements
 
The authors would like to thank Dr Elizabeth Dulmet and Dr Philippe Dartevelle for providing the tumour samples. This work was supported, in part, by a grant from Legs Poix to Y de K.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received 21 June 2005
Accepted 24 October 2005




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