Specific inhibition of mTOR pathway induces anti-proliferative effect and decreases the hormone secretion in cultured pituitary adenoma cells.

There are some evidences that pituitary tumors may be sensitive to the anti-proliferative effects of mammalian target of rapamycin (mTOR) inhibitors, while the mechanism and effects remains unclear, it is necessary to find if a specific mTOR inhibition, including the blocking of both mTOR function and expression, generate any effects on pituitary adenoma cells. The object of this study was to examine if specific inhibition of mTOR induced anti-proliferative effect and decreased the GH and PRL hormones secretion in GH3 and MtT/E pituitary adenoma cells by using a kind of mTOR shRNA lentiviral vector. The in vitro experiments results showed mTOR shRNA transfection robustly reduced the GH3 and MtT/E cells viability in all durations (1-6 days) we performed, also specifically decreased both GH and PRL hormones external secretion in GH3 cells. Further results suggested that specific inhibition of mTOR decreased the hormones secretion through anti-proliferation effects on GH3 cells and reducing the hormones synthesis, but not through affecting the process of hormones secretion. Then we used phosphatidic acid (PA), a kind of mTOR activator, to promote the cell proliferation and GH and PRL hormones secretion in GH3 cells while the effects were blocked by mTOR shRNA transfection. In addition, we examined in vitro effects of PA treatment and mTOR shRNA gene transfection on major proteins expressed in the mTOR pathway in GH3 cells, and confirmed that PA treatment significant increased the protein levels of pmTOR, pS6 K and p4EBP1 in the scramble shRNA group, while the increase of protein levels was blocked by mTOR shRNA gene transfection. Moreover, mTOR shRNA gene transfection definitely inhibited the expression of mTOR and reduced the expression of pmTOR, pS6K and p4EBP1 in either PA or no PA treatment groups. These findings indicated that specific inhibition of mTOR pathway induced anti-proliferative effect and decreased the GH and PRL hormones secretion in cultured pituitary adenoma cells, which may be a novel promising and potential treatment modality for patients with secreting or non-secreting pituitary adenomas.

Recurrent gain-of-function USP8 mutations in Cushing's disease.

Cushing's disease, also known as adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas (PAs) that cause excess cortisol production, accounts for up to 85% of corticotrophin-dependent Cushing's syndrome cases. However, the genetic alterations in this disease are unclear. Here, we performed whole-exome sequencing of DNA derived from 12 ACTH-secreting PAs and matched blood samples, which revealed three types of somatic mutations in a candidate gene, USP8 (encoding ubiquitin-specific protease 8), exclusively in exon 14 in 8 of 12 ACTH-secreting PAs. We further evaluated somatic USP8 mutations in additional 258 PAs by Sanger sequencing. Targeted sequencing further identified a total of 17 types of USP8 variants in 67 of 108 ACTH-secreting PAs (62.04%). However, none of these mutations was detected in other types of PAs (n = 150). These mutations aggregate within the 14-3-3 binding motif of USP8 and disrupt the interaction between USP8 and 14-3-3 protein, resulting in an elevated capacity to protect EGFR from lysosomal degradation. Accordingly, PAs with mutated USP8 display a higher incidence of EGFR expression, elevated EGFR protein abundance and mRNA expression levels of POMC, which encodes the precursor of ACTH. PAs with mutated USP8 are significantly smaller in size and have higher ACTH production than wild-type PAs. In surgically resected primary USP8-mutated tumor cells, USP8 knockdown or blocking EGFR effectively attenuates ACTH secretion. Taken together, somatic gain-of-function USP8 mutations are common and contribute to ACTH overproduction in Cushing's disease. Inhibition of USP8 or EGFR is promising for treating USP8-mutated corticotrophin adenoma. Our study highlights the potentially functional mutated gene in Cushing's disease and provides insights into the therapeutics of this disease.

Transsphenoidal pituitary macroadenomas resection guided by PoleStar N20 low-field intraoperative magnetic resonance imaging: comparison with early postoperative high-field magnetic resonance imaging.

OBJECTIVE: To evaluate the applicability of low-field intraoperative magnetic resonance imaging (iMRI) during transsphenoidal surgery of pituitary macroadenomas. METHODS: Fifty-five transsphenoidal surgeries were performed for macroadenomas (modified Hardy's Grade II-IV) resections. All of the surgical processes were guided by real-time updated contrast T1-weighted coronal and sagittal images, which were acquired with 0.15 Tesla PoleStar N20 iMRI (Medtronic Navigation, Louisville, CO). The definitive benefits as well as major drawbacks of low-field iMRI in transsphenoidal surgery were assessed with respect to intraoperative imaging, tumor resection control, comparison with early postoperative high-field magnetic resonance imaging, and follow-up outcomes. RESULTS: Intraoperative imaging revealed residual tumor and guided extended tumor resection in 17 of 55 cases. As a result, the percentage of gross total removal of macroadenomas increased from 58.2% to 83.6%. The accuracy of imaging evaluation of low-field iMRI was 81.8%, compared with early postoperative high-field MRI (Correlation coefficient, 0.677; P < 0.001). A significantly lower accuracy was identified with low-field iMRI in 6 cases with cavernous sinus invasion (33.3%) in contrast to the 87.8% found with other sites (Fisher's exact test, P < 0.001). CONCLUSION: The PoleStar N20 low-field iMRI navigation system is a promising tool for safe, minimally invasive, endonasal, transsphenoidal pituitary macroadenomas resection. It enables neurosurgeons to control the extent of tumor resection, particularly for suprasellar tumors, ensuring surgical accuracy and safety, and leading to a decreased likelihood of repeat surgeries. However, this technology is still not satisfying in estimating the amount of the parasellar residual tumor invading into cavernous sinus, given the false or uncertain images generated by low-field iMRI in this region, which are difficult to discriminate between tumor remnant and blood within the venous sinus.

A novel gene mutation (1292 deletion) in a Chinese family with cerebral cavernous malformations.

OBJECTIVE: Hereditary cerebral cavernous malformations (CCMs) are characterized by focal abnormalities of small blood vessels in the brain and consequent hemorrhage and seizures. Previous studies of this type of CCM have mainly reported on this disorder in Hispanic and Caucasian cases. Here, we report on hereditary CCM in a Chinese family further characterized by a novel CCM1 gene mutation. METHODS: We investigated a family of 21 members, of whom 3 died and 16 of the survivors became the subjects of this study by brain magnetic resonance imaging. RESULTS: Brain magnetic resonance imaging demonstrated abnormal results in 11 members (69% penetrance), including multiple intracranial lesions in seven cases and single lesions in four cases. The clinical manifestation of CCM was found in these cases. The youngest patient was 4 years old. The remaining 5 members were normal. Nucleotide sequencing analysis of the family member representing the index case and other affected members revealed a deletion frameshift mutation of A and T at nucleotides 1292 and 1293 in exon 13 of the CCM1 gene, which resulted in truncated encoding Krev interaction trapped-1 protein. CONCLUSION: Our results indicated a novel hereditary CCM1 gene mutation of 1292delAT, a finding that may contribute to the clarification of the mechanism of the disease.

Treatment of pituitary adenomas with a transsphenoidal approach.

OBJECTIVE: To evaluate the overall therapeutic effectiveness of transsphenoidal microsurgery for pituitary adenomas and explore the surgical technique, application of new technology, and postoperative follow-up. METHODS: The clinical presentation, imaging features, endocrine examination, pathological types, conditions of operation, postoperative complications, and follow-up of 4050 patients with pituitary adenomas who had undergone transsphenoidal microsurgery from December 1981 to January 2004 were analyzed retrospectively. RESULTS: During the past 6 years, total tumor resection (under microscope) has been achieved in 97.3% of the patients with Hardy Grade I adenomas, 95.2% of Hardy Grade II, 90.4% of Hardy Grade III, and 47.4% of Hardy Grade IV. The percentages of total and subtotal resection achieved extended from 87.6% before 1987 to 96.9% in 2003. CONCLUSION: With the improvement of microsurgical technique and application of novel technology, the indications for transsphenoidal microsurgery for pituitary adenomas were increasingly extended. We believe that more than 90% of pituitary adenomas could be treated by the transsphenoidal approach and totally removed through the microscope. The transnasal perpendicular plate-sphenoid sinus approach will be the dominant mode. Routine postoperative radiotherapy is not required for patients with total resection.

[Identification of a novel inheritable CCM1 gene mutation of 671del AT in a Chinese family with cerebral cavernous malformation].

OBJECTIVE: To investigate the hereditary characters of familial cerebral cavernous malformation (FCCM) and the novel gene mutation in a Chinese family. METHODS: Head MRI examination and clinical neurological check were performed on a Chinese family with one proband of FCCM, female, 27 years old, and 16 family members, 9 males and 12 females, and 19 controls, including patients with sporadic CCM and other diseases and healthy persons. DNA was extracted from the white blood cells of the peripheral blood of the subjects. PCR and DNA direct sequencing were used to detect the mutation in CCM1 gene. RESULTS: Head MRI found 11 FCCM patients in the 16 family members of the proband (69%), the youngest one being 4 years old, including multiple intracranial lesions in 7 patients and single lesion in 4. Relevant clinical manifestations were found in 6 out of the 11 family members. Nucleotide sequence analysis of the proband and other affected family members revealed a deletion frameshift mutation of A and T at nucleotides (nt) 671 and 672 in exon 13 of the CCM1 gene, resulting in truncated encoding KRIT1 protein. No mutation was detected in the healthy family members and the controls. CONCLUSION: A novel inheritable CCM1 gene mutation of 671del AT has been found in patients with FCCM.

[The clinical evaluation of patients with pituitary adenomas undergone transsphenoidal microsurgery].

OBJECTIVE: To evaluate the overall effect of transsphenoidal microsurgery for pituitary adenomas in recent 5 years and to discuss the surgical technique, application of new technology and postoperative follow-up results. METHODS: The clinical presentation, image characteristics, endocrinal findings, pathological types, tumor removal percentage, postoperative complication and follow-up of 1 462 patients with pituitary adenomas who underwent the transsphenoidal microsurgery from 1997 to 2002 were analysed retrospectively. RESULTS: Total rate of tumor removal for the patients achieved 97.0% in the patients with Hardy I adenomas, 95.2% with Hardy II, 90.5% with Hardy III, and 47.4% with Hardy IV respectively. A significant postoperative improvement both in clinical symptoms and endocrinal parameters was achieved. The tumor recurrence rate was 0.3%. CONCLUSIONS: With the improvement of microsurgical technique and application of novel technology, the indication of transsphenoidal microsurgery for pituitary adenomas was increasingly extended. Endoscope and(/or) neuronavigation-assisted microsurgery via transsphenoidal approach should be of the first choice for the treatment of pituitary adenomas. The routine postoperative radiotherapy is not required for patients with total tumor removal.