Impaired Antiviral Responses to Extracellular Double-Stranded RNA and Cytosolic DNA, but Not to Interferon-α Stimulation, in TRIM56-Deficient Cells

The physiologic function of tripartite motif protein 56 (TRIM56), a ubiquitously expressed E3 ligase classified within the large TRIM protein family, remains elusive. Gene knockdown studies have suggested TRIM56 as a positive regulator of the type I interferon (IFN-I) antiviral response elicited via the Toll-like receptor 3 (TLR3) and cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathways, which detect and respond to danger signals—extracellular double-stranded (ds) RNA and cytosolic dsDNA, respectively. However, to what extent these pathways depend on TRIM56 in human cells is unclear. In addition, it is debatable whether TRIM56 plays a part in controlling the expression of IFN-stimulated genes (ISGs) resulting from IFN-I based antiviral treatment. In this study, we created HeLa-derived TRIM56 null cell lines by gene editing and used these cell models to comprehensively examine the impact of endogenous TRIM56 on innate antiviral responses. Our results showed that TRIM56 knockout severely undermined the upregulation of ISGs by extracellular dsRNA and that loss of TRIM56 weakened the response to cytosolic dsDNA. ISG induction and ISGylation following IFN-α stimulation, however, were not compromised by TRIM56 deletion. Using a vesicular stomatitis virus-based antiviral bioactivity assay, we demonstrated that IFN-α could efficiently establish an antiviral state in TRIM56 null cells, providing direct evidence that TRIM56 is not required for the general antiviral action of IFN-I. Altogether, these data ascertain the contributions of TRIM56 to TLR3- and cGAS–STING-dependent antiviral pathways in HeLa cells and add to our understanding of the roles this protein plays in innate immunity.

Increased migration and motility in XIAP-null cells mediated by the C-RAF protein kinase

The product encoded by the X-linked inhibitor of apoptosis (XIAP) gene is a multi-functional protein which not only controls caspase-dependent cell death, but also participates in inflammatory signalling, copper homeostasis, response to hypoxia and control of cell migration. Deregulation of XIAP, either by elevated expression or inherited genetic deletion, is associated with several human disease states. Reconciling XIAP-dependent signalling pathways with its role in disease progression is essential to understand how XIAP promotes the progression of human pathologies. In this study we have created a panel of genetically modified XIAP-null cell lines using TALENs and CRISPR/Cas9 to investigate the functional outcome of XIAP deletion. Surprisingly, in our genetically modified cells XIAP deletion had no effect on programmed cell death, but instead the primary phenotype we observed was a profound increase in cell migration rates. Furthermore, we found that XIAP-dependent suppression of cell migration was dependent on XIAP-dependent control of C-RAF levels, a protein kinase which controls cell signalling pathways that regulate the cytoskeleton. These results suggest that XIAP is not necessary for control of the apoptotic signalling cascade, however it does have a critical role in controlling cell migration and motility that cannot be compensated for in XIAP-knockout cells.

Double adenomas of the pituitary reveal distinct lineage markers, copy number alterations, and epigenetic profiles

Purpose Pituitary adenoma (PA) constitutes the third most common intracranial neoplasm. The mostly benign endocrine lesions express no hormone (null cell PA) or the pituitary hormone(s) of the cell lineage of origin. In 0.5–1.5% of surgical specimens and in up to 10% of autopsy cases, two or three seemingly separate PA may coincide. These multiple adenomas may express different hormones, but whether or not expression of lineage-restricted transcription factors and molecular features are distinct within multiple lesions remains unknown. Methods Searching the data bank of the German Pituitary Tumor Registry 12 double pituitary adenomas with diverse lineage were identified among 3654 adenomas and 6 hypophyseal carcinomas diagnosed between 2012 and 2020. The double adenomas were investigated immunohistochemically for expression of hormones and lineage markers. In addition, chromosomal gains and losses as well as global DNA methylation profiles were assessed, whenever sufficient material was available (n = 8 PA). Results In accordance with the literature, combinations of GH/prolactin/TSH–FSH/LH adenoma (4/12), GH/prolactin/TSH–ACTH adenoma (3/12), and ACTH–FSH/LH adenoma (3/12) were observed. Further, two out of 12 cases showed a combination of a GH/prolactin/TSH adenoma with a null-cell adenoma. Different expression pattern of hormones were confirmed by different expression of transcription factors in 11/12 patients. Finally, multiple lesions that were molecularly analysed in 4 patients displayed distinct copy number changes and global methylation pattern. Conclusion Our data confirm and extend the knowledge on multiple PA and suggest that such lesions may origin from distinct cell types.

The Antitumor Effect of Capsaicin on Prostate Cancer Cells Depends on AMPK Activation Through LKB1 Kinase and TRPV1 Receptor

Recently, natural compounds and their derivatives have been reported to have anti-cancer effects. Herein, we investigated the mechanism whereby the natural vanilloid capsaicin exerts anti-tumor effects on prostate cancer cells. We found that capsaicin activates the AMP-activated kinase (AMPK) and promotes cell death in the LKB1 expressing prostate cancer cell lines LNCaP and PC3 but not in the LKB1-null cell line DU-145. Capsaicin treatment stimulated LKB1 phosphorylation and activated AMPK in LKB1 expressing cells. In addition, overexpression of LKB1 by lentiviral infection in DU-145 cells, induced capsaicin-triggered AMPK activation and apoptosis while LKB1 silencing in LNCaP and PC3 cells, increased capsaicin-promoted cell death. Capsaicin-induced LKB1 phosphorylation, was dependent on the transient receptor potential cation channel subfamily V member 1 (TRPV1), since TRPV1 knocked down by shRNA, abolished LKB1 and AMPK phosphorylation and inhibited apoptosis. Altogether, our results show that capsaicin affect AMPK activity in an LKB1- and TRPV1-dependent fashion, linking TRPV1 with cell fate. These data also suggest that capsaicin may be a rational therapeutic option for prostate tumors.

Morpho-Molecular Metabolic Analysis and Classification of Human Pituitary Gland and Adenoma Biopsies Based on Multimodal Optical Imaging

Pituitary adenomas count among the most common intracranial tumors. During pituitary oncogenesis structural, textural, metabolic and molecular changes occur which can be revealed with our integrated ultrahigh-resolution multimodal imaging approach including optical coherence tomography (OCT), multiphoton microscopy (MPM) and line scan Raman microspectroscopy (LSRM) on an unprecedented cellular level in a label-free manner. We investigated 5 pituitary gland and 25 adenoma biopsies, including lactotroph, null cell, gonadotroph, somatotroph and mammosomatotroph as well as corticotroph. First-level binary classification for discrimination of pituitary gland and adenomas was performed by feature extraction via radiomic analysis on OCT and MPM images and achieved an accuracy of 88%. Second-level multi-class classification was performed based on molecular analysis of the specimen via LSRM to discriminate pituitary adenomas subtypes with accuracies of up to 99%. Chemical compounds such as lipids, proteins, collagen, DNA and carotenoids and their relation could be identified as relevant biomarkers, and their spatial distribution visualized to provide deeper insight into the chemical properties of pituitary adenomas. Thereby, the aim of the current work was to assess a unique label-free and non-invasive multimodal optical imaging platform for pituitary tissue imaging and to perform a multiparametric morpho-molecular metabolic analysis and classification.

Estradiol Augments the Production and Actions of Polymorphonuclear Cells to Promote Lymphangioleiomyomatosis (LAM) Progression

Affecting almost exclusively women, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by slowly growing, estrogen-sensitive metastatic smooth muscle cell-like adenomas that result in cystic lung changes and loss of pulmonary function. LAM tumors are caused by mutations in either TSC1 or TSC2 genes that induces defective inhibition of the mTORC1 pathway, leading to increased mTORC1 activity and augmented cell proliferation. We have previously reported that estrogen ablation in our uterine-specific Tsc2 knockout mouse, which grows tumors with characteristic LAM features and lung colonization potential, effects notable regression of tumors. Thus, estrogen is required for to maintain heightened mTORC1 activity and LAM-like tumor progression. Interestingly, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol in vitro, suggesting that estradiol may act elsewhere—in mTORC1 independent manner—in vivo to promote LAM progression. Flow cytometry revealed large numbers of Ly-6Cint Ly-6Ghigh myeloid cells—polymorphonuclear cells or PMNs—in the blood and myometrial tumors of our uterine-specific Tsc2-null mice. Accordingly, we found that Tsc2-null tumors required PMNs for normal disease progression, as Gr-1 (Ly-6C/Ly-6G) depletion or inhibition of PMN recruitment reduced tumor growth. Therefore, we hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting PMN production in the bone marrow and actions in the tumor microenvironment. Using bone marrow cultures, we found that estradiol is indeed a potent inducer of PMN production. This effect occurs equally in both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ERα; knockout mice, we showed that ERα; is necessary for promoting a PMN fate for myeloid progenitors. Additionally, we have evidence implicating estrogen in the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. Overall, these data suggest that estradiol maybe facilitating crosstalk in LAM tumors, directly stimulating tumor cells while also promoting the production and actions of PMNs, which in turn promote tumor growth.

Somatotroph Adenomas have a Predilection to Invade the Cavernous Sinus and Resection of the Medial Wall of the Cavernous Sinus Offers the Highest Potential for Biochemical Remission in Acromegaly

Recurrence and remission rates vary widely among different histological subtypes of pituitary adenoma. Invasion of the medial wall of the cavernous sinus is a known mechanism that may account for such failed clinical outcomes as its removal has long been considered unattainable. The use of modern endoscopic techniques allows for direct intraoperative evaluation of invasion and resection of the medial wall of the cavernous sinus with low morbidity when performed by highly experienced surgeons. In this retrospective study we evaluated 105 consecutive primary pituitary adenomas operated by a single surgeon including 28 corticotroph, 27 gonadotroph, 24 somatotroph, 15 lactotroph, 5 null-cell, 5 plurihormonal, and 1 dual adenoma; 53 caused hypersecretory syndromes, specifically acromegaly (30), hyperprolactinemia (15) and Cushing’s disease (8). In each case, we performed meticulous intraoperative inspection of the medial wall with its surgical removal when invasion was suspected, regardless of functional status. Medial wall resection was performed in 46% of pituitary adenomas, and 38/48 walls confirmed pathologic evidence of invasion rendering a positive predictive value of intraoperative evaluation of medial wall invasion of 79%. Furthermore, we show for the first time that the rate of medial wall invasion among pathological subtypes is dramatically different. Somatotroph tumors invaded the medial wall much more often than other adenoma subtypes, 83% intraoperatively and 71% histologically, followed by plurihormonal tumors (40%) and gonadotrophs (33%), both with intraoperative positive predictive value of 100%. The least likely to invade were corticotroph, at a rate of 32% intraoperatively and 21% histologically, and null-cell adenomas at 0%. Removal of the medial wall caused no permanent morbidity with no carotid artery injuries and 2 patients with transient diplopia. We report that resecting the medial wall of the cavernous sinus in acromegaly offers the highest potential for biochemical remission with average postoperative day 1 GH levels at 0.96 ug/l and early surgical remission rates at 90% (100% with adjuvant therapy) based on normalization of IGF-1 levels 3 to 6 months after surgery; these results are significantly better than previously reported but longer follow-up is required for definitive conclusions. Our findings may explain the failed biochemical remission rates seen in acromegaly and illustrate the relevance of advanced surgical techniques for successful outcomes in pituitary surgery.

Characterization of Gonadotroph Pituitary Adenomas Based on the Recent 2017 WHO Pituitary Tumor Classification

Introduction: More than 20% of pituitary adenomas are nonfunctional, the majority of which are of gonadotroph origin. Whereas previously, immunohistochemistry of pituitary hormones was used to classify adenoma subtypes, in 2017 the World Health Organization (WHO) reclassified pituitary adenomas using transcription factor expression in addition to immunohistochemistry. With this change, clinically nonfunctional gonadotroph adenomas can be distinguished among: (1) those staining for the transcription factor SF-1 and gonadotropins FSH and/or LH (FSH/LH+), (2) those that stain for SF-1 but not for FSH or LH (FSH/LH- SF1+), and (3) true null cell adenomas. It is unclear whether these three subgroups behave similarly clinically, or if they have distinct manifestations or outcomes. Our aim was to characterize these subgroups in regard to tumor size, recurrence and pituitary insufficiency. Methods: In a retrospective chart review, 71 patients from 2017-2020 who presented to the hospital for transsphenoidal resection of clinically nonfunctioning pituitary adenomas were reviewed. All patients with pituitary adenomas that stained positive for SF-1 and negative for T-PIT and PIT-1, and tumors that were negative for all three transcription factors were evaluated. Those lacking clinical data were excluded. Clinical characteristics examined include: demographics, tumor size, invasion of cavernous sinus, and hormone deficiencies. Results: Of the 71 pituitary tumors, 45% (n=32) stained positive for the beta subunit FSH and/or LH (FSH/LH+) and SF-1, 44% (n=31) stained for SF-1 with negative pituitary hormone stains (FSH/LH- SF1+), and 11% (n=8) were negative for all transcription factors and hormones (true null). All tumors were macroadenomas (>1 cm). While there were >50% males in the FSH/LH+ and FSH/LH- SF1+ groups, in the true null group only 25% of patients were male. Most patients were >50 years old in all 3 groups (81% FSH/LH+, 75% FSH/LH- SF1+, 88% true null). The prevalence of cavernous sinus involvement was 36% in both groups that stained for SF-1, but was 62% in the true null group. Both SF-1+ groups had similar tumor sizes and prevalence of panhypopituitarism (15-21%), but there were more episodes of recurrence since last known follow up in the FSH/LH- SF1+ group (20%), compared to FSH/LH+ tumors (7%). The true null group had ≥50% rates for both panhypopituitarism and recurrence. Conclusions: In this study, we highlighted the category of FSH/LH- SF1+ gonadotroph adenomas and compared these to FSH/LH+ and true null cell tumors. Based on clinical features, FSH/LH- SF1+ gonadotroph adenomas are similar to FSH/LH+ staining pituitary adenomas in regard to age, sex, size, and degree of cavernous sinus invasion, although there were more recurrences in the FSH/LH- SF1+ group. Though less common, our cohort suggests more aggressive tendencies in the true null group compared to SF-1 staining tumors.

Evaluation of Hsp90 and mTOR inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.