Truncated somatostatin receptor variant sst5TMD4 confers aggressive features (proliferation, invasion and reduced octreotide response) to somatotropinomas

Abstract

The GH/IGF1 response of somatotropinomas to somatostatin analogues (SSA) is associated with their pattern of somatostatin receptor (sst1-sst5) expression. Recently, we demonstrated that expression of a truncated sst5-variant (sst5TMD4) can influence the secretory response of somatotropinomas to SSA-therapy; however, its potential relationship with aggressive features (e.g. invasion/proliferation) is still unknown. Here, we show that sst5TMD4 is present in 50% of non-functioning pituitary-adenomas (NFPA) (n = 30) and 89% of somatotropinomas (n = 36), its expression levels being highest in somatotropinomas > > NFPAs > > > normal pituitaries (negligible expression; n = 8). In somatotropinomas, sst5TMD4 mRNA and protein levels correlated positively, and its expression was directly associated with tumor invasiveness (cavernous/sphenoid sinus), and inversely correlated with age and GH/IGF1 reduction after 3-6 months with octreotide-LAR therapy. GNAS+ somatotropinomas expressed lower sst5TMD4 levels. ROC analysis revealed sst5TMD4 expression as the only marker, within all sst-subtypes, capable to predict tumor invasiveness in somatotropinomas. sst5TMD4 overexpression increased cell viability in cultured somatotropinoma (n = 5). Hence, presence of sst5TMD4 associates with increased aggressive features and worse prognosis in somatotropinomas, thereby providing a potentially useful tool to refine somatotropinoma diagnosis, predict outcome of clinical response to SSA-therapy and develop new therapeutic targets.

Keywords: Acromegaly; Invasion; Proliferation; sst5TMD4.

Fig. 1

(A) Median sst5TMD4 mRNA levels in normal pituitaries (NP; n = 8), somatotropinomas (GHomas; n = 36) and non-functioning pituitary adenomas (NFPA; n = 30). (B) Representative IHC images of sst5TMD4 in somatotropinomas. (C) Positive correlation between IHC intensity and percentage of positive cells with sst5TMD4 mRNA levels. (D) Negative correlation between the age of acromegalic patients at diagnosis and sst5TMD4 levels. (A–D) mRNA copy number corrected by a normalization factor (NF) derived from the expression of three control genes (HPRT1, ACTB, and GAPDH).

Fig. 2

(A) Negative correlations between sst2/sst5TMD4 ratio and GH and IGF1 levels after 3 and 6 months postsurgical treatment with octreotide-LAR in acromegalic patients. (B) sst2/sst5TMD4 ratio in pharmacologically controlled and uncontrolled patients with GH secreting adenomas (GHomas). (C) sst5TMD4 expression and sst2/sst5TMD4 ratio in patients with GNAS negative and positive tumors.

Fig. 3

sst5TMD4 expression levels in invasive somatotropinomas [total (cavernous + sphenoid) sinus extension; left, top-panel], as well as in tumors with cavernous (middle, top-panel) or sphenoid (right, top-panel) sinus extension; Receiver operating characteristics (ROC) curve to determine the accuracy of sst5TMD4 receptor as diagnostic test to discriminate between invasive and non-invasive somatotropinomas (bottom-panels).

Fig. 4

Cell viability (24-, 48- and/or 72-h) in primary culture of somatotropinoma transfected with sst5TMD4 (A, n = 5; black columns) and sst5TMD5 (B, n = 3; gray-columns) as compared with mock-transfected control cells (white-columns). Representative validation by qPCR sst5TMD4 (C, black bar) and sst5TMD5 (D, light gray bar) demonstrating an increase in sst5TMD4, but not in sst5 (C and D, dark gray bar), mRNA levels. Data are expressed as percent of mock, set at 100%.