Global gene expression in neuroendocrine tumors from patients with the MEN1 syndrome

Abstract

Background: Multiple Endocrine Neoplasia type 1 (MEN1, OMIM 131100) is an autosomal dominant disorder characterized by endocrine tumors of the parathyroids, pancreatic islets and pituitary. The disease is caused by the functional loss of the tumor suppressor protein menin, coded by the MEN1 gene. The protein sequence has no significant homology to known consensus motifs. In vitro studies have shown menin binding to JunD, Pem, Smad3, NF-kappaB, nm23H1, and RPA2 proteins. However, none of these binding studies have led to a convincing theory of how loss-of-menin leads to neoplasia.

Results: Global gene expression studies on eight neuroendocrine tumors from MEN1 patients and 4 normal islet controls was performed utilizing Affymetrix U95Av2 chips. Overall hierarchical clustering placed all tumors in one group separate from the group of normal islets. Within the group of tumors, those of the same type were mostly clustered together. The clustering analysis also revealed 19 apoptosis-related genes that were under-expressed in the group of tumors. There were 193 genes that were increased/decreased by at least 2-fold in the tumors relative to the normal islets and that had a t-test significance value of p < or = 0.005. Forty-five of these genes were increased and 148 were decreased in the tumors relative to the controls. One hundred and four of the genes could be classified as being involved in cell growth, cell death, or signal transduction. The results from 11 genes were selected for validation by quantitative RT-PCR. The average correlation coefficient was 0.655 (range 0.235-0.964).

Conclusion: This is the first analysis of global gene expression in MEN1-associated neuroendocrine tumors. Many genes were identified which were differentially expressed in neuroendocrine tumors arising in patients with the MEN1 syndrome, as compared with normal human islet cells. The expression of a group of apoptosis-related genes was significantly suppressed, suggesting that these genes may play crucial roles in tumorigenesis in this syndrome. We identified a number of genes which are attractive candidates for further investigation into the mechanisms by which menin loss causes tumors in pancreatic islets. Of particular interest are: FGF9 which may stimulate the growth of prostate cancer, brain cancer and endometrium; and IER3 (IEX-1), PHLDA2 (TSS3), IAPP (amylin), and SST, all of which may play roles in apoptosis.

Figure 1

Histogram showing the frequency of genes being expressed at levels between 50 and 7875 (arbitrary expression units).

Figure 2

Coefficient of variation (CV) of genes being expressed at levels between 50 and 6000. For each gene expressed at an average level of 50 or above, the CV was computed for the group of 8 tumors, for the group of 4 normals, and for the group of all 12 tumors and normals. As the lower limit of expression was increased, the number of genes represented in the CV decreased: there were 12,000 genes with expression levels of 50 or more, but only a few genes with expression levels of 6,500 or more.

Figure 3

Clustering of tumors and normals according to overall gene expression patterns. The predominant type of hormone expression (Table 3) is noted for each tumor/normal specimen.

Figure 4

Clustering of apoptosis-related genes in tumors (T) and normals (N). Pink indicates strong, white indicates moderate, and blue indicates weak expression.

Figure 5

The expression levels of 4 apoptosis-related genes are shown by GeneChip and quantitative RT-PCR: a) IER3; b) IAPP; c) SST; d) PHLDA2. Normals (N) and tumors (T) are shown. Solid bars represent GeneChip and open bars represent Q-PCR results.

Figure 6

FGF9 expression levels in tumors (T) and normals (N) by GeneChip and quantitative RT-PCR. Solid bars represent GeneChip and open bars represent Q-PCR results.