Hi, I'm Rainier Alvarez. I am a medical student at the Herbert Gordon College of Medicine at Florida International University, and I am an NIH Medical Research Scholar in the Neuroendocrine Surgery Unit of the Surgical Neurology Branch under the mentorship of Dr. Prashant Chidi-Voina. And today I'll be talking to you about Cushing's disease pituitary adenomas and how they regulate noxiprotein to evade cellular death. I have no disclosures to report. And I'd like to acknowledge the NIH Medical Research Scholars Program for allowing me the opportunity to spend a year at the NIH, to pursue my research interests, and receive the funding through the fellowship. So Cushing's disease is a state of hypercortisolemia caused by a benign autonomous pituitary microadenoma. These patients have an increased mortality, as well as suffer from multiple comorbidities. And the first line of treatment currently for Cushing's disease is transphenoidal surgery. However, these pituitary microadenomas are very small and difficult to detect, and thus make surgery much more difficult, as well as causing multiple patients to suffer from surgical failure, which is a lack of clinical remission after surgery. And some patients also suffer from recurrence many years after surgery. So what other options do we have for these patients? Second line treatment for Cushing's disease currently is radiation therapy, such as stereotactic radiosurgery. And using stereotactic radiosurgery, however, has shown to have a lower success rate for patients achieving clinical remission, as well as increasing the risk of patients having panhypopituitarism. And the third line treatment for these patients are medications, such as cabergoline, pasireotide, which are approved for Cushing's disease. However, these medications have their own profile of adverse effects, as well as also having a lower success rate for patients achieving clinical remission. You may have noticed that for all of these treatments, they're all directed at the symptomatology or physical removal of the tumor. None of these treatment modalities are actually targeting the specific cause of these tumors or the tumorigenesis related to Cushing's disease. So when I arrived at the Chiriboyna lab, I wanted to study and further understand the biology of these Cushing's disease tumors and how they undergo this tumorigenic reprogramming. Thankfully, at our lab, which focuses on the clinical and direct translational aspects of Cushing's disease, I had the opportunity to pursue this. So currently, what we do know is that these tumors arise from pituitary corticotrophs, and around 30% of these tumors acquire a somatic USP8A mutation or gene, point mutation in the USP8A gene that leads to tumor formation. However, we don't know which event or what is the cause for the remaining 70% of these tumors. We do know that all these tumors share unregulated tumor growth, ACTH over secretion, and a different metabolic profile than that seen in normal pituitary corticotrophs. We also know that in general, across various different benign and malignant human cancers, tumorigenesis is related to dysregulated apoptosis. So today, I'd like to talk about how dysregulated apoptosis may be a factor in Cushing's disease tumor formation. So what do we know about altered apoptosis in various pituitary adenomas? So literature has shown that in an article that focused on non-functional pituitary adenomas, they found a 47-fold decrease of the PMA-IP1 transcript when comparing the adenomas to autopsy-derived normal human pituitary glands. The gene PMA-IP1 is the gene that encodes the NOXA protein, which is a protein that is involved in the intrinsic apoptotic pathway. Another article showed that PMA-IP1 transcript had a 66.1-fold decrease in the adenomas when compared to autopsy-derived human normal pituitary gland. So the NOXA protein is a mediator of apoptosis in damaged cells and prevents tumor formation when it's functional and is present. So let's talk about NOXA a bit more. So as you see here, NOXA is a BH3 protein involved in the intrinsic apoptotic cascade. It inhibits the BCL2 family, specifically NOXA is a pro-apoptotic sensitizer by inhibiting the BCL2 family, which then allows BaxanBac to allow for mitochondrial outer membrane permeabilization, cytochrome c release, and activation of the cytoplasmic caspases, which would then cause apoptosis and prevention of tumor formation. So to further understand the tumorogenic reprogramming in Cushing disease adenomas and the possibility of dysregulated apoptosis being a factor, I was interested in analyzing the transcriptome of Cushing disease adenomas a bit further. So at the NIH, we have one of the world's largest Cushing disease patient populations, and this allowed us to collect surgically-derived humans and genetic pairs of adenomas and normal pituitary gland, as well as additional adenoma-only samples, and analyze them all together via pooled RNA-seq. So these syngeneic pairs are unique and essential to our study. They consist of adenoma and surrounding normal pituitary gland samples that are taken from the same patient at the same time during surgery. They're identified and resected using careful microsurgical dissection to preserve the histology and the cell morphology of these samples. Having the opportunity of analyzing these paired samples freshly right after surgery allows us to preserve the transcriptome within these cells and prevent any transcriptional changes that may occur after death or during autopsy-procured samples. So from this analysis, we found 74 targets that had a linear fold change of 1.5 or greater in adenomas when compared to the surrounding normal pituitary gland. Of these 74 transcripts, we noticed that PMA-IP1, the gene from Noxa, was one of them. Specifically, it showed a four-fold increase in the Cushing disease adenomas when compared to surrounding normal pituitary gland. However, we didn't see this when we analyzed non-functional adenoma and prolactinoma syngeneic pairs. This brought up an interesting point, though. How is the transcript for this pro-epitotic protein upregulated, however, these tumors are not undergoing any apoptosis? On the contrary, they're tumors, they're continuing to grow. So we decided to analyze the protein expression levels of Noxa in these tumors. So using various Cushing disease adenomas, growth hormone-secreting adenoma, and non-functional adenoma, surgically-derived tissues of patients cared for at the NIH Clinical Center, we were able to subject the immunohistochemistry to quantitative measurements using digital image analysis after thresholding, and we noticed that Noxa was suppressed in 8 out of 10 Cushing disease samples of adenomas compared to their adjacent normal pituitary gland. However, this Noxa suppression wasn't seen in the growth hormone-secreting or non-functional adenomas. And these findings were verified and confirmed by our board-certified and trained neuropathologist. We then decided to examine the in vitro Noxa protein expression in primary cell cultures that were grown from surgically-derived patient adenomas, and we noticed that in the Cushing disease cells, there was a variable expression of Noxa protein that didn't correlate with our findings of the RNA sequencing data. So this led us to consider the possibility of post-translational modifications that may be targeting the Noxa protein. So in the literature, it has been described how Noxa does undergo proteasomal degradation in various tumors, and how this post-survival adaptation can even allow certain tumors to develop chemotherapeutic resistance. So we decided to apply this to our current samples and see if this was a possibility of the phenomenon we've been seeing. So using the adenoma samples that we derived from surgical specimens of patients cared at the clinical center, we first began treating the cells with various compounds. We have in the first lane towards the far left, these cells were just treated with DMSO, which is the vehicle used to reconstitute the different compounds. We then treated the cells with EGF for 24 hours, expecting to see a drop in Noxa levels, considering that EGF will stimulate cell growth and diminish apoptosis. However, we did not see a change. Cells treated with Gfinitive for six hours did show an increase in Noxa, as we would expect, as Gfinitive does induce cell apoptosis. And we also treated the cells with specific proteasomal inhibitor, Bordezumab, where we saw the greatest and most robust increase in Noxa expression. Using a less selective proteasomal inhibitor, Orlistat, also showed an increase in Noxa, however, to a lesser degree. We were able to replicate these findings in a separate adenoma sample, where we also saw a robust increase in Noxa, specifically using Bordezumab and Orlistat. Using these findings, it allowed us to conclude that proteasomal post-translational degradation of Noxa might represent a post-survival adaptation in Cushing disease adenomas. And if this is so, the use of proteasomal inhibitors, such as Bordezumab, may elevate Noxa protein levels in vivo and induce apoptosis in Cushing disease adenomas, providing an alternative therapy that targets the cause of these tumors. I'd like to thank the members of my lab, my mentor Dr. Chittiboyna, Dr. Heiss, my advisor, and Dr. Mondal for helping me coordinate and plan many of these experiments, as well as those members of the Neuroendocrine Surgery Unit and the Surgical Neurology Branch. Thank you.

Rainier Alvarez

Cushing's disease

pituitary adenomas

NOXA protein

proteasomal inhibitors