Neuroendocrine-like Cells Research Articles

Abstract 4697: Surface localization of enolase-1 (ENO-1) as an attractive theranostic target in docetaxel-resistant neuroendocrine-like prostate cancer cells

Abstract Prostate cancer (PCa) is American men's second most common cancer. Although taxane-based chemotherapy is the last line of defense for Metastatic Castration Resistant Prostate Cancer (mCRPC), it invariably fails due to chemoresistance. The protein-specific membrane antigen (PSMA) has been an effective target for the imaging and therapy of mCRPC. Although PSMA radioligand therapy (PSMA-RLT) is a theranostics option for men with advanced PCa, about 30% have a limited response due to neuroendocrine-like PCa (NEPC), which lacks PSMA expression. Enolase (ENO) is a promising theranostic alternative due to its cell surface localization in many human tumors. We observed that chemosensitive PCa cell lines expressing NEPC markers express both ENO-1 and ENO-2; however, docetaxel-resistant NEPC-like cells only express ENO-1 and have a metabolic vulnerability due to the loss of ENO-2. We hypothesize that ENO-1 is expressed on the surface of NEPC-like cell lines and can be targeted with small molecule inhibitors (SMIs) that could potentially be used as theranostics agents. Additionally, we have observed changes in the expression and localization of ENO-1 in NEPC-like cell lines under different glucose concentrations. Our data show that under high glucose conditions, found on metabolically active metastatic tumors, ENO-1 is highly expressed on the cell surface, making it a promising candidate target for theranostics. However, low glucose conditions inhibit the activity of the c-MYC oncogene resulting in ENO-1 downregulation and upregulation of MBP1, the small splice variant of ENO1 that blocks the transcriptional activity of c-MYC. Our efforts to confirm ENO-1 surface expression on NEPC-like cell lines entailed using confocal microscopy, cell fractionation analysis followed by Western blotting, and Flow cytometric cell surface staining. We also evaluated the cytotoxicity of SMIs designed to target ENO-1 in chemoresistant NEPC-like cell lines using MTT viability assays, clonogenic assays, and Hoffman Modulation microscopy imaging. Our long-term goal is to identify an alternative treatment for patients with NEPC by establishing ENO-1 as a novel theranostics target. Citation Format: Krystal Rubi Santiago Torres, Carlos A. Casiano, Frankis Almaguel, Alfonso Duran, Kristen Whitley. Surface localization of enolase-1 (ENO-1) as an attractive theranostic target in docetaxel-resistant neuroendocrine-like prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4697.

Abstract 1493: Secretory function of neuroendocrine-like prostate cancer cell enhances therapy resistance and metastatic potential of adjacent adenocarcinoma

Abstract Background: Castration-resistant prostate cancer (CRPC) is advanced non curable disease associated with poor prognosis and survival n patients. Androgen receptor pathway inhibitors (ARSIs) are used to treat advanced prostate cancer, but in majority of patients it progresses by developing resistance to AR-directed therapies and is referred to as CRPC. Adenocarcinoma cells in CRPC tumor often undergoes lineage switch and become neuroendocrine-like cancer cells (NEPC) with small-cell histology, characterized by the loss of Tp53 and Rb1 genes. NEPC cells are frequently present as scattered foci along with prostatic adenocarcinomas and are secretory in nature. Objective: The primary objective is to determine how these NEPC cells influence surrounding adenocarcinoma and aids in disease progression. Methods: We performed Mass spec and cytokine array experiments from NEPC conditioned media to determine its secretory phenotype. We orthotopically implanted NEPC and adeno cells in the nude mice and performed colony formation assay on flow sorted adeno cells from the tumor. We also checked the effect of NEPC on the adeno cells and TME components. Results: Our analysis of the RNA-seq data from SU2C-PCF cohort and NE-like cells revealed that functions related to secretion were significantly enriched in NE-like PCa. NE-like cells gain the ability to secrete neuropeptides, growth factors, and cytokines to establish cell-cell communication. This secretory function is tightly regulated by the Neuropilin2 (NRP2) axis. We studied for the effect of NE-like cells on the adenocarcinoma cells in the mice tumor model and found that the presence of NE-like cells facilitated the adenocarcinoma cells to acquire aggressive characteristics. This could be due to both direct and/or through the modulation of the tumor microenvironment (TME). The direct effect could be by enhancing the therapy resistant ability of the surrounding adenocarcinoma, and the indirect effect is through the tumor microenvironment especially through neurons and macrophages that promote metastatic potential. Conclusions: Overall, our results prove that NE-like cancer cells influence the adjacent adenocarcinoma cells directly by making them resistant to treatment and indirectly make them more metastatic by utilizing macrophage-neuron axis. Citation Format: Juhi Mishra, Sanika Bodas, Sreyashi Bhattacharya, Dipanwita Das, Sheeraz UnNazir, Samikshan Dutta, Kaustubh Datta. Secretory function of neuroendocrine-like prostate cancer cell enhances therapy resistance and metastatic potential of adjacent adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1493.

Diagnostic dilemma in a patient with history of medullary thyroid carcinoma and abnormal serum liver enzymes; a case report with six years follow up

BackgroundMedullary thyroid carcinoma (MTC) is a neuroendocrine tumor that originates from parafollicular C-cells. Calcitonin (Ctn) and carcinoembryonic antigen (CEA) are useful biomarkers for monitoring MTC cases.Case presentationHere, we describe a 48-year-old woman, who presented in 2014 with bilateral thyroid nodules. Report of fine needle aspiration was suspicious for MTC; initial laboratory evaluation showed serum Ctn level of 1567 pg/mL. After excluding type 2 multiple endocrine neoplasia syndrome clinically, total thyroidectomy and neck lymph node dissection were performed. The final histopathological diagnosis was right lobe MTC with neither vascular invasion nor lymph node involvement. On regular follow-up visits, Ctn and CEA levels have been undetectable, and repeated cervical ultrasonographic exams were unremarkable from 2014 to 2021. As liver enzymes became elevated in 2016, the patient was further evaluated by a gastroenterologist. Abdominopelvic ultrasonography revealed a coarse echo pattern of the liver parenchyma with normal bile ducts. A liver fibroscan showed a low fibrosis score (7kPa). The patient was recommended to use ursodeoxycholic acid. According to the progressive rise of liver enzymes with a cholestatic pattern in October 2020, a liver biopsy was performed that showed tiny nests of neuroendocrine-like cells with a background of primary biliary cholangitis (PBC). Immunohistochemical stainings were positive for chromogranin A (CgA), and synaptophysin and negative for Ctn, CEA, and thyroglobulin. Further imaging investigations did not reveal any site of a neuroendocrine tumor in the body. Considering normal physical exam, imaging findings, as well as normal serum levels of Ctn, CEA, CgA, and procalcitonin, the patient was managed as a PBC.ConclusionIn follow-up of a patient with MTC, we reported progressively increased liver enzymes with a cholestatic pattern. Liver biopsy revealed nests of neuroendocrine-like cells with a background of PBC, the findings that might suggest acquiring neuroendocrine phenotype by proliferating cholangiocytes.

Palisading Adenocarcinoma: A Morphologically Unique Salivary Gland Tumor With a Neuroendocrine-like Appearance and a Predilection for the Sublingual Glands of Women.

Adenocarcinoma, not otherwise specified (NOS) is a heterogenous group of salivary gland tumors that likely contains distinct tumors that have not yet been characterized. Indeed, in recent years, cases previously diagnosed as adenocarcinoma, NOS have been recategorized into novel tumor designations such as secretory carcinoma, microsecretory adenocarcinoma, and sclerosing microcystic adenocarcinoma. We sought to describe a distinctive, hitherto-undescribed salivary gland tumor encountered in the authors' practices. Cases were pulled from the surgical pathology archives of the authors' institutions. Histologic, immunohistochemical, and clinical findings were tabulated, and targeted next-generation sequencing was performed on all cases. Nine cases were identified, arising in 8 women and 1 man ranging from 45 to 74 years (mean, 56.7y). Seven tumors (78%) arose in the sublingual gland, while 2 (22%) arose in the submandibular gland. The cases shared a distinctive morphologic appearance. They were biphasic, with ducts scattered among a predominant polygonal cell with round nuclei, prominent nucleoli, and pale eosinophilic cytoplasm. These cells were arranged as trabeculae and palisaded as pseudorosettes around hyalinized stroma and vessels, resembling a neuroendocrine tumor. Four of the cases were well-circumscribed, while the remaining 5 showed infiltrative growth including perineural invasion in 2 (22%) and lymphovascular invasion in 1 (11%). Mitotic rates were low (mean, 2.2/10HPFs); necrosis was absent. By immunohistochemistry, the predominant cell type was strongly positive for CD56 (9 of 9) and variably positive for pan-cytokeratin (AE1/AE3) (7 of 9) with patchy S100 (4 of 9), but negative for synaptophysin (0 of 9) and chromogranin (0 of 9), while the ducts were strongly positive for pan-cytokeratin (AE1/AE3) (9 of 9) and CK5/6 (7 of 7). Next-generation sequencing did not reveal any fusions or obvious driver mutations. All cases were resected surgically, with external beam radiation also done in 1 case. Follow-up was available in 8 cases; there were no metastases or recurrences after 4 to 160 months (mean, 53.1mo). A dual population of scattered ducts with a predominance of CD56-positive neuroendocrine-like cells characterizes a unique salivary gland tumor which is often encountered in the sublingual glands of women, for which we propose the term "palisading adenocarcinoma." Although the tumor was biphasic and had a neuroendocrine-like appearance, it lacked convincing immunohistochemical evidence of myoepithelial or neuroendocrine differentiation. Although a subset showed unequivocally invasive growth, this tumor appears to behave in an indolent manner. Moving forward, recognition of palisading adenocarcinoma and its separation from other salivary adenocarcinomas, NOS will facilitate a better understanding of the characteristics of this previously unrecognized tumor.

PRMT5 promotes chemotherapy-induced neuroendocrine differentiation in NSCLC.

In response to therapeutic treatments, cancer cells can exhibit a variety of resistance phenotypes including neuroendocrine differentiation (NED). NED is a process by which cancer cells can transdifferentiate into neuroendocrine-like cells in response to treatments, and is now widely accepted as a key mechanism of acquired therapy resistance. Recent clinical evidence has suggested that non-small cell lung cancer (NSCLC) can also transform into small cell lung cancer (SCLC) in patients treated with EGFR inhibitors. However, whether chemotherapy induces NED to confer therapy resistance in NSCLC remains unknown. We evaluated whether NSCLC cells can undergo NED in response to chemotherapeutic agents etoposide and cisplatin.By Knock-down of PRMT5 or pharmacological inhibition of PRMT5 to identify its role in the NED process. We observed that both etoposide and cisplatin can induce NED in multiple NSCLC cell lines. Mechanistically, we identified protein arginine methyltransferase 5 (PRMT5) as a critical mediator of chemotherapy-induced NED. Significantly, the knock-down of PRMT5 or pharmacological inhibition of PRMT5 suppressed the induction of NED and increased the sensitivity to chemotherapy. Taken together, our results suggest that targeting PRMT5 may be explored as a chemosensitization approach by inhibiting chemotherapy-induced NED.

JNJ-64619178 radiosensitizes and suppresses fractionated ionizing radiation-induced neuroendocrine differentiation (NED) in prostate cancer.

Radiation therapy (RT) is a standard treatment regimen for locally advanced prostate cancer; however, its failure results in tumor recurrence, metastasis, and cancer-related death. The recurrence of cancer after radiotherapy is one of the major challenges in prostate cancer treatment. Despite overall cure rate of 93.3% initially, prostate cancer relapse in 20-30% patients after radiation therapy. Cancer cells acquire radioresistance upon fractionated ionizing radiation (FIR) treatment, eventually undergo neuroendocrine differentiation (NED) and transform into neuroendocrine-like cells, a mechanism involved in acquiring resistance to radiation therapy. Radiosensitizers are agents that inhibit the repair of radiation-induced DNA damage. Protein arginine methyltransferase 5 (PRMT5) gets upregulated upon ionizing radiation treatment and epigenetically activates DNA damage repair genes in prostate cancer cells. In this study, we targeted PRMT5 with JNJ-64619178 and assessed its effect on DNA damage repair gene activation, radiosensitization, and FIR-induced NED in prostate cancer. γH2AX foci analysis was performed to evaluate the DNA damage repair after radiation therapy. RT-qPCR and western blot were carried out to analyze the expression of DNA damage repair genes. Clonogenic assay was conducted to find out the surviving fraction after radiation therapy. NED was targeted with JNJ-64619178 in androgen receptor (AR) positive and negative prostate cancer cells undergoing FIR treatment. JNJ-64619178 inhibits DNA damage repair in prostate cancer cells independent of their AR status. JNJ-64619178 impairs the repair of ionizing radiation-induced damaged DNA by transcriptionally inhibiting the DNA damage repair gene expression and radiosensitizes prostate, glioblastoma and lung cancer cell line. It targets NED induced by FIR in prostate cancer cells. JNJ-64619178 can radiosensitize and suppress NED induced by FIR in prostate cancer cells and can be a potential radiosensitizer for prostate cancer treatment.

Neuropilin-2 axis in regulating secretory phenotype of neuroendocrine-like prostate cancer cells and its implication in therapy resistance.

SUMMARYNeuroendocrine (NE)-like tumors secrete various signaling molecules to establish paracrine communication within the tumor milieu and to create a therapy-resistant environment. It is important to identify molecular mediators that regulate this secretory phenotype in NE-like cancer. The current study highlights the importance of a cell surface molecule, Neuropilin-2 (NRP2), for the secretory function of NE-like prostate cancer (PCa). Our analysis on different patient cohorts suggests that NRP2 is high in NE-like PCa. We have developed cell line models to investigate NRP2’s role in NE-like PCa. Our bioinformatics, mass spectrometry, cytokine array, and other supporting experiments reveal that NRP2 regulates robust secretory phenotype in NE-like PCa and controls the secretion of factors promoting cancer cell survival. Depletion of NRP2 reduces the secretion of these factors and makes resistant cancer cells sensitive to chemotherapy in vitro and in vivo. Therefore, targeting NRP2 can revert cellular secretion and sensitize PCa cells toward therapy.

Targeting Protein Arginine Methyltransferase 5 Suppresses Radiation-induced Neuroendocrine Differentiation and Sensitizes Prostate Cancer Cells to Radiation.

Prostate cancer remains the second leading cause of cancer death among American men. Radiotherapy is a potentially curative treatment for localized prostate cancer, and failure to control localized disease contributes to the majority of prostate cancer deaths. Neuroendocrine differentiation (NED) in prostate cancer, a process by which prostate adenocarcinoma cells transdifferentiate into neuroendocrine-like (NE-like) cells, is an emerging mechanism of resistance to cancer therapies and contributes to disease progression. NED also occurs in response to treatment to promote the development of treatment-induced neuroendocrine prostate cancer (NEPC), a highly aggressive and terminal stage disease. We previously demonstrated that by mimicking clinical radiotherapy protocol, fractionated ionizing radiation (FIR) induces prostate cancer cells to undergo NED in vitro and in vivo. Here, we performed transcriptomic analysis and confirmed that FIR-induced NE-like cells share some features of clinical NEPC, suggesting that FIR-induced NED represents a clinically relevant model. Furthermore, we demonstrated that protein arginine methyltransferase 5 (PRMT5), a master epigenetic regulator of the DNA damage response and a putative oncogene in prostate cancer, along with its cofactors pICln and MEP50, mediate FIR-induced NED. Knockdown of PRMT5, pICln, or MEP50 during FIR-induced NED and sensitized prostate cancer cells to radiation. Significantly, PRMT5 knockdown in prostate cancer xenograft tumors in mice during FIR prevented NED, enhanced tumor killing, significantly reduced and delayed tumor recurrence, and prolonged overall survival. Collectively, our results demonstrate that PRMT5 promotes FIR-induced NED and suggests that targeting PRMT5 may be a novel and effective radiosensitization approach for prostate cancer radiotherapy.

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

NEFL is overexpressed and it modulates invasion and migration in neuroendocrine-like PC3-ML2 prostate cancer cells.

Prostate cancer clinical outcomes are varied, from non-aggressive asymptomatic to lethal aggressive neuroendocrine forms which represent a critical challenge in the management of the disease. The neurofilament light ( NEFL ) is proposed to be a tumor suppressor gene. Studies have shown that expression of the gene is decreased in various cancers. We have used quantitative RT-PCR, immunoblotting, methylation specific PCR, siRNA knockdown followed by migration/invasion assays to determine associations between NEFL expression and disease phenotype in a panel of prostate cells. We demonstrate that NEFL is overexpressed and it modulates invasion and migration in PC3-ML2 prostate cancers cells which have an aggressive neuroendocrine-like phenotype.

Regulation of Neuroendocrine-like Differentiation in Prostate Cancer by Non-Coding RNAs.

Neuroendocrine prostate cancer (NEPC) represents a variant of prostate cancer that occurs in response to treatment resistance or, to a much lesser extent, de novo. Unravelling the molecular mechanisms behind transdifferentiation of cancer cells to neuroendocrine-like cancer cells is essential for development of new treatment opportunities. This review focuses on summarizing the role of small molecules, predominantly microRNAs, in this phenomenon. A published literature search was performed to identify microRNAs, which are reported and experimentally validated to modulate neuroendocrine markers and/or regulators and to affect the complex neuroendocrine phenotype. Next, available patients’ expression datasets were surveyed to identify deregulated microRNAs, and their effect on NEPC and prostate cancer progression is summarized. Finally, possibilities of miRNA detection and quantification in body fluids of prostate cancer patients and their possible use as liquid biopsy in prostate cancer monitoring are discussed. All the addressed clinical and experimental contexts point to an association of NEPC with upregulation of miR-375 and downregulation of miR-34a and miR-19b-3p. Together, this review provides an overview of different roles of non-coding RNAs in the emergence of neuroendocrine prostate cancer.

Abstract 1408: PlexinD1 drives enzalutamide-resistant neuroendocrine prostate cancer development and progression

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer (PC) arising as a consequence of more potent targeting of the androgen receptor (AR) pathway in castration-resistant prostate cancer (CRPC). Recently, the transition from CRPC to NEPC occurs more frequently due to the widespread use of highly potent antiandrogens such as enzalutamide (ENZ). This study sought to investigate the functional role of PlexinD1 in ENZ-induced NEPC and dissect the underlying molecular mechanism. Methods: The PlexinD1 expression levels and its association with neuroendocrine differentiation were determined in publicly available multi-omics datasets and clinical specimens. Human PC stable cell lines with augmentation or silencing of PlexinD1 expression were established and assayed for cell proliferation, colony formation and neuroendocrine marker gene expression by Western blotting and RT-qPCR analyses. Results: PlexinD1 expression was elevated in hormone-refractory PC compared to hormone-sensitive PC and further in NEPC compared to CRPC, which was paralleled by a positive correlation between PlexinD1 expression and NEPC score and an inverse relationship of PlexinD1 levels with AR score, by a survey of multiple independent clinical datasets. The association of PlexinD1 with different subtypes and disease status of PC will also be explored in our own patient cohorts by quantitative immunohistochemical analysis. The PlexinD1 mRNA levels were induced in androgen-dependent LNCaP cells grown in hormone-depleted media for 3 days and in C4-2B cells treated with 10 µM ENZ for 5 days. PlexinD1 was also increased in ENZ-resistant C4-2B (C4-2BENZR) cells at both mRNA and protein levels. shRNA-mediated knockdown of PlexinD1 in neuroendocrine-like PC cells, including C4-2BENZR, 22Rv1, PC-3 and DU145, suppressed cell proliferation by up to more than 50%, which was corroborated by decreased colony formation. PlexinD1 silencing in C4-2BENZR and 22Rv1 cells led to reduced expression of the NE marker chromogranin A (CHGA). The effects of PlexinD1 on NEPC tumor growth and neuroendocrine marker expression will be further examined in xenograft mouse models. Collectively, our data indicate that PlexinD1 plays an essential role supporting the acquisition of a neuroendocrine phenotype in PC cells and driving NEPC cell survival and growth. Conclusion: Our study suggests that PlexinD1 promotes the progression of CRPC to NEPC under ENZ pressure and subsequent emergence of resistance to ENZR possibly through induction of neuroendocrine traits, which provides new insights into further developing PlexinD1 as a new biomarker and therapeutic target in NEPC. Funding provided by Department of Defense PCRP grant W81XWH-19-1-0279, NIH/NCI grant R37CA233658, and the WSU start-up fund (to B.W.) Citation Format: Jing Wang, Jingjing Li, Boyang Wu. PlexinD1 drives enzalutamide-resistant neuroendocrine prostate cancer development and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1408.

Increased Expression of AKT3 in Neuroendocrine Differentiated Prostate Cancer Cells Alters the Response Towards Anti-Androgen Treatment.

Simple SummaryMetastatic castration-resistant prostate cancer is still untreatable, and patients have a very short life expectancy after diagnosis. One factor that makes metastatic castration-resistant prostate cancer so aggressive and difficult to treat is neuroendocrine differentiation of prostate carcinoma cells. We already showed that neuroendocrine differentiation of LNCaP cells results in increased AKT3 expression. The aim of this study was to demonstrate the role of AKT3 in neuroendocrine differentiation. Therefore, the previously obtained in vitro data were validated and extended to tissue from patient with neuroendocrine prostate cancer, where we show the presence of AKT3 in neuroendocrine cells. Furthermore, we demonstrate the oncogenic consequences of an increased AKT3 expression including inactivation of apoptotic proteins and a potential role of some miR-17 family members, negatively regulating AKT3, in neuroendocrine differentiation.Patients with advanced prostate carcinoma are often treated with an androgen deprivation therapy but long-term treatment can result in a metastatic castration-resistant prostate cancer. This is a more aggressive, untreatable tumor recurrence often containing areas of neuroendocrine differentiated prostate cancer cells. Using an in vitro model of NE-like cancer cells, it could previously be shown that neuroendocrine differentiation of LNCaP cells leads to a strong deregulation of mRNA and miRNA expression. We observe elevated RNA and protein levels of AKT Serine/Threonine Kinase 3 (AKT3) in neuroendocrine-like LNCaP cells. We used prostate resections from patients with neuroendocrine prostate cancer to validate these results and detect a co-localization of neuroendocrine marker genes with AKT3. Analysis of downstream target genes FOXO3A and GSK3 strengthens the assumption AKT3 may play a role in neuroendocrine differentiation. Overexpression of AKT3 shows an increased survival rate of LNCaP cells after apoptosis induction, which in turn reflects the significance in vivo or for treatment. Furthermore, miR-17, −20b and −106b, which are decreased in neuroendocrine-like LNCaP cells, negatively regulate AKT3 biosynthesis. Our findings demonstrate AKT3 as a potential therapeutic target and diagnostic tool in advanced neuroendocrine prostate cancer and a new mRNA–miRNA interaction with a potential role in neuroendocrine differentiation of prostate cancer.

Mechanisms of castration resistant prostate cancer formation and progression through neuroendocrine differentiation

Normal prostate tissues consist mainly of epithelial cells, including secretory epithelial cells, basal cells, and neuroendocrine cells, and of mesenchymal cells, including smooth muscle cells and fibroblasts. The mechanisms leading to castration resistant prostate cancer (CRPC) are complex and diverse, but most involve neuroendocrine differentiation. In fact, during the development of prostate cancer, some of the tumor cells transform into neuroendocrine-like cells. This transition is a main underlying mechanism of CRPC formation.

Abstract PO-059: Understanding the lineage of metaplastic tuft cells in the progression of PDA

Abstract Pancreatic ductal adenocarcinoma (PDA) is unique as it is marked by a dramatic increase in the collagen deposition tumor microenvironment. This microenvironment in pancreatic tumors is composed of immune cells and reactive fibroblasts, with the tumor cells themselves comprising only a small proportion of the overall mass. Nearby normal epithelial tissue is also reactive, undergoing a transdifferentiation event known as acinar-to-ductal metaplasia (ADM), where normal acinar cells are replaced by metaplastic duct-like structures. Collectively neoplastic and metaplastic epithelium orchestrate the immune and fibrotic response, and understanding how the pancreatic epithelia communicate to the microenvironment may lead to more effective clinical treatments. Metaplastic tuft cells (MTCs) are a specialized subset of the epithelium that has the potential to drive tumor progression through communication with the microenvironment and modulate PDA progression. Also known as solitary chemosensory cells, tuft cells were first discovered in rodent luminal surfaces more than 60 years ago. They are characterized by the “tuft” of microvilli reaching into the lumen and, only recently, studies have started to determine the role of normal tuft cells in different organs. These studies determined that tuft cells have different roles depending on the organ in which they reside. Studies have shown that tuft cells are not present in a normal pancreas. MTCs are only present in the pancreas in PanINs during PDA progression in both humans and mice. Furthermore, the population of MTCs in the pancreas disappears as PDA progresses into invasive carcinoma. MTCs also express other markers outside of the taste sensing proteins, including POU2f3/SKN-1alpha, and DCLK1. We know little about the role of MTCs in the pancreas, but prior studies have suggested their role as a progenitor cell during PDA. However, these studies do not exclusively mark MTCs during their genesis in a progressive model of PDA due to a lack of mouse model as well as the complexity of culturing them ex vivo. We have generated a unique mouse model to drive lineage tracing of MTCs during PDA, and I have preliminary data to suggest that tuft cells can transdifferentiate into neuroendocrine-like cells during PDA progression. Neuroendocrine (NE) cells are found in aggressive cancers such as prostate and small cell lung cancer. They are characterized by markers such as Synaptophysin (SYP), as well as an upregulation of MYC which has been found to be more aggressive in different cancers. Rosalie Sears, at the Oregon Health and Science University, a specialist of MYC function in PDA, has shown that NE cells in the pancreas not only originate from acinar cells, where MTCs originate but whose presence is associated with poor survival and resistance to treatment in PDA. She has also shown that MYC plays a key role in the regulation of NE cells in PDA. Through my dissertation research, I will investigate tuft to NE-like transdifferentiation and its role in PDA progression. Citation Format: Daniel J. Salas-Escabillas, Megan Hoffman, Howard C. Crawford. Understanding the lineage of metaplastic tuft cells in the progression of PDA [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-059.

Paragangliome de la queue de cheval : à propos d’une série de 9 cas

The paraganglioma of the cauda equina is a rare tumor, the diagnosis is morphological and the immunohistochemistry provides a definite diagnosis. The objective of our study is to specify the clinical signs, radiological and associated pathological criteria and to compare our data with those of the literature. This is a retrospective study of nine cases of paragangliomas of the cauda equina diagnosed in our department from 2003 to 2018. The median age of the patients was 50 years-old with a male predominance (sex ratio: 3,5/1). All patients had preoperative magnetic resonance imaging (MRI) and surgery to remove the tumor. The diagnosis was performed after HES (Hematoxylin Eosin Saffron) and immunohistochemical sections examination. Radiculalgia was the chief symptom of these tumors. MRI showed an oval lesion uniformly enhanced by Gadolinium in the eight patients whose records were available. Histologically, the tumors had a lobular and trabecular pattern with neuroendocrine-like cells and a rich vascularization. By immunohistochemistry, the cells expressed chromogranin, synaptophysin and CD56. Paragangliomas of the cauda equina are rare, benign tumors. Except for cases of secreting tumors, the preoperative diagnosis is difficult. MRI is useful and may reveal radiological features suggestive of these tumors. However, it is rare for the diagnosis to be made before surgery. The diagnosis is established by histological examination and immunohistochemical techniques must be used to confirm the diagnosis. The paragangliomas of the cauda equina are well encapsulated tumors whose complete excision is curative. When the excision is incomplete, treatment with radiotherapy is recommended. Long-term clinical and radiological monitoring is recommended because of the slow evolution of the tumor and the potential for recurrence.

The regulation of HAS3 by miR-10b and miR-29a in neuroendocrine transdifferentiated LNCaP prostate cancer cells

Prostate cancer (PCa) is the second most common type of cancer in male worldwide. During neuroendocrine transdifferentiation (NETD), PCa cells are able to differentiate into androgen-independent neuroendocrine-like (NE-like) tumor cells, which are associated with reduced survival rates in PCa patients. The molecular processes underlying NETD have not been clarified yet, but miRNAs could play a potential role. MiRNAs are short, single-stranded, non-coding RNA molecules that regulate gene expression post-transcriptionally by binding to the 3′-untranslated region (3′UTR) of their target mRNAs. This study aimed to explore the possible relevance and function of the transmembrane Hyaluronan Synthase 3 (HAS3) and miR-10b as well as miR-29a during NETD.Here, we validated a repression of HAS3 and an induction of miR-10b and miR-29a by quantitative real-time PCR after NETD. HAS3 was predicted as a new target gene for both miRNAs, which was verified by Reporter Gene Assays and Western Blotting. Functional analyses revealed an inhibiting effect of HAS3 on cell proliferation and migration in LNCaP cells, whereas miR-10b showed no impact. Furthermore, HAS3 increased the colony forming ability, while miR-10b diminished it. These results might give a hint on the role of miR-10b and HAS3 during NETD of PCa cells.

Cytoplasmic Her2/neu Immunohistochemical Staining in Breast Cancer; From a Molecular Point of View.

The most widely used guideline for the breast cancer biomarker assessment and reporting (the 2013 ASCO/CAP guideline) does not state the unusual occurrence of cytoplasmic Her2/neu staining (1, 2). We recently encountered a T2N1Mx ductal adeno-carcinoma which consisted of two dissimilar tumor cell populations. The more prominent population (75% of tumor cells) was made up of sheets of neuroendocrine-like cells (NEL) and the other tumor cell population had a usual adenocarcinomatous histomorphology (UAC) (Fig. 1A). The NEL was ER+ (clone 073), PgR-(clone 636), 40% ki67 with distinct dot-like cytoplasmic Her2 staining (clone CB11) which is considered as negative regarding the current guidelines. The UAC was ER+, PgR+, 20% ki67, and Her2 negative (Fig. 1A-C). Moreover, NEL did not react with either chromogranin or synaptophysin, but it expressed neuron-specific enolase (NSE). Dual color Her2/neu chromogenic in situ hybridization probes (chromogenic ISH) established that both components were not amplified for this oncoprotein gene (Fig. 1D).

Long-Term Engraftment Promotes Differentiation of Alveolar Epithelial Cells from Human Embryonic Stem Cell Derived Lung Organoids.

Human embryonic stem cell (hESC) derived 3D human lung organoids (HLOs) provide a promising model to study human lung development and disease. HLOs containing proximal or/and immature distal airway epithelial cells have been successfully generated in vitro, such as early staged alveolar type 2 (AT2) cells (SPC+/SOX9+) and immature alveolar type 1 (AT1) cells (HOPX+/SOX9+). When HLOs were transplanted into immunocompromised mice for further differentiation in vivo, only few distal epithelial cells could be observed. In this study, we transplanted different stages of HLOs into immunocompromised mice to assess whether HLOs could expand and mature in vivo. We found that short-term transplanted HLOs contained lung progenitor cells (NKX2.1+, SOX9+, and P63+), but not SPC+ AT2 cells or AQP5+ AT1 cells. Meanwhile, long-term engrafted HLOs could differentiate into lung distal bipotent progenitor cells (PDPN+/SPC+/SOX9+), AT2 cells (SPC+, SPB+), and immature AT1 cells (PDPN+, AQP5-). However, HLOs at late in vitro stage turned into mature AT1-like cells (AQP5+/SPB-/SOX9-) in vivo. Immunofluorescence staining and transmission electron microscopy (TEM) results revealed that transplanted HLOs contained mesenchymal cells (collagen I+), vasculature (ACTA2+), neuroendocrine-like cells (PGP9.5+), and nerve fiber structures (myelin sheath structure). Together, these data reveal that hESC-derived HLOs would be useful for human lung development modeling, and transplanted HLOs could mimic lung organ-like structures in vivo by possessing vascular network and neuronal network.

The deregulation of miR-17/CCND1 axis during neuroendocrine transdifferentiation of LNCaP prostate cancer cells.

Prostate carcinoma contain foci of neuroendocrine transdifferentiation, resulting in an increase of androgen-independent neuroendocrine-like (NE) tumor cells, whose number significantly correlates with tumor aggressiveness and thus lower survival rate. Neuroendocrine transdifferentiation of prostate cancer cells and a potential role of miRNAs within this process are poorly understood. MicroRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression. The aim of this project was to identify new genes and miRNAs involved in neuroendocrine transdifferentiation. LNCaP prostate cancer cells were differentiated to NE-like cancer cells and microarray analyses were performed. Microarray results have been validated for the eight most deregulated mRNAs and microRNAs via qRT-PCR and analyzed with different algorithms to predict new targets for deregulated microRNAs. The induced CyclinD1 gene could be validated as new target gene for the repressed miR-17 family containing miR-17, miR-20a, miR-20b, miR-106a and miR-106b via reporter gene assays and Western Blot. Functional analysis of miR-17 family shows a high influence on cell proliferation, colony forming ability and apoptosis in LNCaP cells. Our data demonstrate wide changes in mRNA and microRNA expression during neuroendocrine transdifferentiation of LNCaP cells and confirm new mRNA-miRNA interactions with potential roles in NE-transdifferentiation of prostate carcinoma.