Low Levels of Raf Kinase Inhibitory Protein in Growth Hormone-Secreting Pituitary Adenomas Correlate with Poor Response to Octreotide Treatment

Research Institute for Internal Medicine, Section of Endocrinology, University of Oslo, Oslo N-0027, Norway.
Journal of Clinical Endocrinology &amp Metabolism (Impact Factor: 6.21). 05/2008; 93(4):1211-6. DOI: 10.1210/jc.2007-2272
Source: PubMed


Excessive GH production by pituitary tumors causes acromegaly. Medical treatment of acromegaly with somatostatin analogs (SMSs), like octreotide, is well established, but the clinical effect is variable. One mechanism for octreotide effect is inhibition of the MAPK signaling pathway after binding to the G protein-coupled somatostatin receptor. Nonphosphorylated Raf kinase inhibitory protein (RKIP) binds to and inhibits Raf1 kinase, and thereby attenuates MAPK signaling, whereas phosphorylated RKIP inhibits G protein receptor internalization and degradation due to inhibition of G protein receptor kinase 2. Objective: Our objective was to study RKIP levels in pituitary somatotroph adenomas, and relate them to clinical characteristics and response to octreotide treatment in patients with acromegaly.
RKIP level was analyzed by Western blot of proteins extracted from somatotroph tumors frozen a short time after surgery in 51 patients with active acromegaly. An acute somatostatin test was performed in 46 of the patients, and in 21 the IGF-I level before and 6 months after SMS treatment was available.
The adenoma RKIP level correlated significantly to both the acute and the long-term octreotide responses on serum levels of GH and IGF-I, respectively. In multiple regression analyses, the RKIP level was a significant determinant for both the GH reduction in the acute test and the IGF-I reduction after approximately 6 months.
The RKIP level in somatotroph adenomas seems to be important for the clinical effect of SMS treatment, in which low levels of RKIP correlate to poor clinical response to SMSs.

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    • "A reduced response to octreotide/lanreotide has been associated with several findings. Besides the rare sst gene mutations [19] and a single report of antibodies to octreotide [20], high initial GH concentrations [21], a sparse granulation pattern in somatotropinomas [22] [23], low levels of the E-cadherin expression in tumor cytoplasm [24], low levels of Raf kinase inhibitory protein [25], presence of an AIP mutation [26], high values of Ki-67 [27], younger age of patients [28] and male gender [29] have also been described (see [30] for review). However, it is still unclear if these are directly causes for reduced response to treatment with octreotide/lanreotide. "
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    ABSTRACT: Background Somatotropinomas have unique “fingerprints” of somatostatin receptor (sst) expression, which are targets in treatment of acromegaly with somatostatin analogues (SSAs). However, a significant expression of sst is not always related to the biochemical response to SSAs. Headache is a common complaint in acromegaly and considered a clinical marker of disease activity. SSAs are reported to have an own analgesic effect, but the sst involved are unknown. Patients and methods We investigated sst expression in two acromegalic patients with severe headache and no biochemical effects of octreotide, but a good response to pasireotide. We searched the literature for determinants of biochemical and analgesic effects of SSAs in somatotropinomas. Results Case 1 had no biochemical or analgesic effects of octreotide, a semi-selective SSA, but a rapid and significant effect of pasireotide, a pan-SSA. Case 2 demonstrated discordance between analgesic and biochemical effects of octreotide, in that headache disappeared, but without biochemical improvement. In contrast, pasireotide normalized insulin-like growth factor 1. Both adenomas were sparsely granulated and had strong membranous expressions of sst2a in 50–75% and sst5 in 75–100% of tumor cells. The truncated sst5 variant TMD4 (sst5TMD4) showed expression in 20–57% of tumor cells. Conclusions A poor biochemical response to octreotide may be associated with tumor expression of a truncated sst5 variant, despite abundant sst2a expression, suggesting an influence from variant sst5 on common sst signaling pathways. Furthermore, unrelated analgesic and biochemical effects of SSAs supported a complex pathogenesis of acromegaly-associated headache. Finally, assessment of truncated sst5 in addition to full length sst could be important for a choice of postoperative SSA treatment in somatotropinomas.
    Growth hormone & IGF research: official journal of the Growth Hormone Research Society and the International IGF Research Society 10/2015; 25(5):262–267. DOI:10.1016/j.ghir.2015.07.003 · 1.41 Impact Factor
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    • "Studies have demonstrated the altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas [79], Gsα and Giα mutations in clinically nonfunctioning pituitary adenomas [80], and an H-ras mutation in a single aggressive prolactinoma or metastases from pituitary carcinomas [81]. Recent studies demonstrate that an overexpression of B-Raf mRNA and protein is a feature of nonfunctional pituitary adenomas; that overactivity highlights an overactivity of the Ras-B-Raf-MAP kinase pathway to promote pituitary tumorigenesis [82], and that the low levels of Raf kinase inhibitory protein (RKIP) in a GH-pituitary adenoma correlate with poor clinical response to somatostatin analog therapy because RKIP can bind to and inhibit Raf1 kinase to attenuate MAPK signaling [83]. The antiproliferative effect of somatostatin analogs involves the upregulation of p27 and downregulation of the MAPK pathway in human somatotrophinomas [84]. "
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    ABSTRACT: We obtained a series of pituitary adenoma proteomic expression data, including protein-mapping data (111 proteins), comparative proteomic data (56 differentially expressed proteins), and nitroproteomic data (17 nitroproteins). There is a pressing need to clarify the significant signaling pathway networks that derive from those proteins in order to clarify and to better understand the molecular basis of pituitary adenoma pathogenesis and to discover biomarkers. Here, we describe the significant signaling pathway networks that were mined from human pituitary adenoma proteomic data with the Ingenuity pathway analysis system. The Ingenuity pathway analysis system was used to analyze signal pathway networks and canonical pathways from protein-mapping data, comparative proteomic data, adenoma nitroproteomic data, and control nitroproteomic data. A Fisher's exact test was used to test the statistical significance with a significance level of 0.05. Statistical significant results were rationalized within the pituitary adenoma biological system with literature-based bioinformatics analyses. For the protein-mapping data, the top pathway networks were related to cancer, cell death, and lipid metabolism; the top canonical toxicity pathways included acute-phase response, oxidative-stress response, oxidative stress, and cell-cycle G2/M transition regulation. For the comparative proteomic data, top pathway networks were related to cancer, endocrine system development and function, and lipid metabolism; the top canonical toxicity pathways included mitochondrial dysfunction, oxidative phosphorylation, oxidative-stress response, and ERK/MAPK signaling. The nitroproteomic data from a pituitary adenoma were related to cancer, cell death, lipid metabolism, and reproductive system disease, and the top canonical toxicity pathways mainly related to p38 MAPK signaling and cell-cycle G2/M transition regulation. Nitroproteins from a pituitary control related to gene expression and cellular development, and no canonical toxicity pathways were identified. This pathway network analysis demonstrated that mitochondrial dysfunction, oxidative stress, cell-cycle dysregulation, and the MAPK-signaling abnormality are significantly associated with a pituitary adenoma. These pathway-network data provide new insights into the molecular mechanisms of human pituitary adenoma pathogenesis, and new clues for an in-depth investigation of pituitary adenoma and biomarker discovery.
    BMC Medical Genomics 04/2010; 3(1):13. DOI:10.1186/1755-8794-3-13 · 2.87 Impact Factor
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    ABSTRACT: Raf Kinase Inhibitor Protein (RKIP) was originally described as an inhibitor of the Ras-Raf-MEK-ERK pathway, exerting its action by the physical inhibition of the interaction of Raf with MEK. It has subsequently been shown to play important roles in a number of other signalling pathways, including the NFκB pathway and in the stability of the mitotic spindle. Not surprisingly given that it impacts on many important signalling pathways RKIP levels have been shown to be important in the progression of a number of different cancers. RKIP expression is lost or decreased in a number of common human cancers and decreased still further in tumour metastases. One of the tumours in which RKIP is downregulated is colorectal cancer (CRC). Importantly it has been shown that not only is RKIP depleted in tumour tissue when compared with normal tissue but that the level of RKIP within a tumour is inversely correlated with the likelihood of metastatic relapse and with patient prognosis. Although we already have a number of very good prognostic indicators in CRC, one group of patients for whom new prognostic indicators would be useful are patients with Dukes B CRC. These are patients with locally advanced but non-metastatic disease and at present there is no firm consensus on their correct post-operative management. Therefore we set out to examine whether RKIP is a useful prognosticator in this particular group using a tissue microarray (TMA) with samples from over 200 patients with Dukes B CRC. The analysis revealed a strong inverse correlation between RKIP levels and disease specific survival. Moreover, in a multivariate analysis RKIP emerged as an independent prognostic indicator along with lympho-vascular invasion and peritoneal invasion, two well-known and powerful prognosticators. This allowed for the generation of a simple prognostic index, using information from the different independent indicators, allowing for improved patient risk stratification. This led us to examine whether RKIP could also function as a predictive marker in CRC. To do this we again used a TMA, this time consisting of a much larger cohort of patients across the whole range of tumour stages. The results confirmed the prognostic utility of RKIP and indicated that patients whose tumours have low levels of RKIP may derive a greater benefit from chemotherapy than those patients whose tumours have high levels, although this result did not reach statistical significance. In the second part of the thesis I have examined the effect of RKIP in previously characterised mouse models of CRC. To do this I have used a germline RKIP knockout mouse and in the first instance crossed it to the APC580S mouse. In this mouse APC is lost conditionally within the intestine and liver. RKIP knockout did not have any effect on the rate of tumourigenesis or on the invasiveness of tumours in this model. However, in the setting of acute homozygous deletion of APC, RKIP knockout resulted in a decrease in apoptoses in the small intestine and an increase in aberrant mitotic activity in the liver. To follow this up I have examined the effect of RKIP knockout in a mouse model of superficially invasive CRC, specifically to see if RKIP knockout can promote invasive and metastatic behaviour. In this model the APC580S mouse is crossed to mice which conditionally express oncogenic KRas. Although RKIP knockout did not result in an increase in invasive tumours in this model there was a shift in tumour location from the small intestine to the colon. This shift appeared to be due, at least in part to an increase in chromosomal instability in the tumours. The final aim of the thesis was to develop a mouse model of CRC which more closely recapitulates the late stages of the human disease, specifically invasion and metastasis. To do this we have crossed the APC580S mouse with either a conditional p53 knockout or with a mouse that conditionally expresses a point mutation of p53 (p53R172H). In human tumours the majority of abnormalities of p53 are point mutations that result in the production of mutant protein that accumulates in tumour cells. There is evidence that this mutant protein may have oncogenic properties beyond the simple loss of normal p53 protein function. Therefore we have also used this model to study the differing effects of p53 loss and point mutation in CRC. We found that mice homozygous for p53 deletion (p53fl/fl) and those expressing a single copy of the mutant allele with loss of the second copy (p53R172H/fl) developed invasive tumours with nearly 100% penetrance and indeed metastasis was observed. Remarkably, although mice that were heterozygous for p53 deletion (p53fl/+) only rarely developed invasive tumours almost 100% of mice expressing a single copy of the mutant allele (p53R172H/+) developed invasive tumours. We went on to show that the increase in invasion seen in this model is related to an increase in Wnt signalling, which is associated with increased expression of pro-invasive Wnt targets such as fascin. We also showed a novel pro-invasive role for ARF in this process. This is also an excellent model of Dukes B CRC and therefore the ideal model to test the effect of RKIP deletion on invasion and metastasis. These studies led us to examine the differences in effect between knockout and mutant p53 in another tumour model. In this we used a novel model of the aggressive tumour pleomorphic rhabdomyosarcoma to demonstrate that mutant p53 can both promote both tumourigenesis and metastasis more potently than p53 knockout. These studies have demonstrated the value of RKIP in the clinically important Dukes B CRC population and shown its possible utility as a predictive marker in this group. Although we have not seen an effect of RKIP knockout in traditional mouse models of CRC we have developed a novel model which closely recapitulates Dukes B CRC and may be useful in elucidating the effect of RKIP knockout. We have also used this model to gain novel insights into the invasive process, in particular into the role played by mutant p53.
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