GHRH Receptor Research Articles

Characterization and regulation of the neonatal growth hormone surge.

High neonatal growth hormone (GH) secretion has been described in several species. However, the neuroendocrine mechanisms behind this surge remain unknown. Thus, the pattern of postnatal GH secretion was investigated in mice and rats. Blood GH levels were very high on postnatal day (P)1 and progressively decreased until near zero by P17 in C57BL/6 mice without sex differences. This pattern was similar to that observed in rats, except that female rats showed higher GH levels on P1 than males. In comparison, follicle-stimulating hormone exhibited higher secretion in females during the first three weeks of life. Hypothalamic Sst mRNA and somatostatin neuroendocrine terminals in the median eminence were higher in P20/P21 mice than in newborns. Knockout mice for GH-releasing hormone (GHRH) receptor showed no GH surge, whereas knockdown mice for the Sst gene displayed increased neonatal GH peak. Leptin deficiency caused only minor effects on early-life GH secretion. GH receptor ablation in neurons or the entire body did not affect neonatal GH secretion, but the subsequent reduction in blood GH levels was attenuated or prevented by these genetic manipulations, respectively. This phenotype was also observed in knockout mice for the insulin-like growth factor-1 (IGF-1) receptor in GHRH neurons. Moreover, glucose-induced hyperglycemia overstimulated GH secretion in neonatal mice. In conclusion, GH surge in the first days of life is not regulated by negative feedback loops. However, neonatal GH secretion requires GHRH receptor, and is modulated by somatostatin and blood glucose levels, suggesting that this surge is controlled by hypothalamic-pituitary communication.

Antagonist of Growth Hormone-Releasing Hormone Receptor MIA-690 Suppresses the Growth of Androgen-Independent Prostate Cancers

The development of resistance remains the primary challenge in treating castration-resistant prostate cancer (CRPC). GHRH receptors (GHRH-R), which are coupled to G-proteins (GPCRs), can mediate EGFR transactivation, offering an alternative pathway for tumour survival. This study aimed to evaluate the effects of the GHRH-R antagonist MIA-690, in combination with the EGFR inhibitor Gefitinib, on cell viability, adhesion, gelatinolytic activity, and the cell cycle in advanced prostate cancer PC-3 cells. The findings demonstrate a synergistic effect between MIA-690 and Gefitinib, leading to the inhibition of cell viability, adhesion, and metalloprotease activity. Cell cycle analysis suggests that both compounds induce cell cycle arrest, both individually and in combination. Furthermore, similar effects of the GHRH-R antagonist MIA-690 combined with Gefitinib were observed in PC-3 tumours developed by subcutaneous injection in athymic nude mice 36 days post-inoculation. These results indicate that combined therapy with a GHRH-R antagonist and an EGFR inhibitor exerts a stronger antitumor effect compared to monotherapy by preventing transactivation between EGFR and GHRH-R in CRPC.

Growth hormone-releasing hormone and cancer.

The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting the synthesis and release of growth hormone (GH), stimulates the proliferation of human normal and malignant cells by binding to GHRH-receptor (GHRH-R) and its main splice variant, SV1. Both GHRH and GHRH-Rs are expressed in various cancers, forming a stimulatory pathway for cancer cell growth; additionally, SV1 possesses ligand independent proliferative effects. Therefore, targeting GHRH-Rs pharmacologically has been proposed for the treatment of cancer. Various classes of synthetic GHRH antagonists have been developed, endowed with strong anticancer activity in vitro and in vivo, in addition to displaying anti-inflammatory, antioxidant and immune-modulatory functions. GHRH antagonists exert indirect effects by blocking the pituitary GH/hepatic insulin-like growth factor I (IGF-I) axis, or directly inhibiting the binding of GHRH on tumor GHRH-Rs. Additionally, GHRH antagonists block the mitogenic functions of SV1 in tumor cells. This review illustrates the main findings on the antitumor effects of GHRH antagonists in experimental human cancers, along with their underlying mechanisms. The development of GHRH antagonists, with reduced toxicity and high stability, could lead to novel therapeutic agents for the treatment of cancer and inflammatory diseases.

8649 Ablation of Leptin Receptors in Somatotropes Impacts Transcriptomic Plasticity in the Pou1f1 Lineage of Female Pituitary Cells

Abstract Disclosure: T.K. Miles: None. A.K. Odle: None. S. Byrum: None. A. Lagasse: None. A. Haney: None. V.G. Ortega: None. A. Herdman: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. Anterior pituitary somatotropes produce growth hormone (GH) to optimize body composition, receiving tropic signals from leptin about the metabolic state. When leptin receptors are ablated in somatotropes, GH proteins, serum GH and GHRH receptors are reduced. Unexpectedly, POU1F1, Prolactin, and TSH-beta proteins are also reduced in pituitaries of Somatotrope LEPR-null females (mutants), suggesting a tropic role for leptin in differentiation. To test this hypothesis, single cell RNA-seq analysis compared the pituitary transcriptome of 6-month-old control and mutant female mice housed under thermoneutral conditions (28°C). Following scRNA-seq, cells were clustered computationally with the use of Seurat; all cell types were identified in separate UMAP clusters. Mutants showed increases in cell numbers in somatotrope (4.7X) and Pou1f1 (5.7x) clusters, but no change in thyrotrope or lactotrope clusters. In LEPR-null female somatotropes, the 516 differentially expressed genes (DEGs; adj p<0.05) included upregulated Pomc, Prl, Lhb, Tshb and Cga (Log2FC+0.5-1.7) and downregulated Pou1f1, Ghrhr, and Gh (Log2FC -0.5—1.5). In the lactotrope cluster from mutants, Pomc, Cga, and Gh were upregulated (Log2FC+1.1-1.5) and Pou1f1 and Prl were downregulated. The thyrotrope cluster was unchanged. Prl, Pomc, and Gh were upregulated in the Pou1f1 cluster. The down regulation of Gh, Pou1f1, and Ghrhr in somatotropes and Prl in lactotropes correlates with the lower serum levels of GH and Prl in mutant females. However, the upregulated DEGs do not correlate with increased secretory activity suggesting that the multihormonal cells are not fully functional. This was confirmed with Ingenuity Pathway Analysis (IPA) showing downregulation of canonical pathways involved in secretion in mutant somatotropes. Unexpectedly, mutant females exposed to a HFD (60% fat) for 16 weeks (+16.6g) showed a reversal of the changes in somatotrope cell numbers and multihormonal expression seen in control fed mutants. The increase in genes belonging to IPA canonical cytoplasmic translation pathways in mutant somatotropes were also completely reversed following a HFD. Furthermore, the downregulated secretory/signaling pathways seen in control fed mutant female somatotropes and lactotropes (endocytosis, vesicle transport, Gaq, GnRH) were upregulated modestly following a HFD. The high serum leptin in the HFD fed mutant mice may activate signaling pathways in cells still expressing LEPR. Collectively, these results confirm a role for leptin in maintaining differentiated somatotropes. They also suggest that leptin may limit expansion of multihormonal progenitor cells. The decreased multihormonal expression and protein synthesis in lactotrope clusters from mutant mice exposed to a HFD are also seen in lactotropes from control mice on a HFD, which suggests that a HFD may compromise pituitary plasticity. Presentation: 6/3/2024

7765 Exploring the Synergistic Effect of GHRH Receptor Agonist MR-409 and Incretin-based Therapies in Type 1 Diabetes Using a Low-dose Streptozotocin Model

Abstract Disclosure: M. Gonzalez Medina: None. R. Louzada: None. V. Pita-Grisanti: None. M. Blandino: None. T. Cui: None. W. Sha: Owner/Co-Owner; Self; WS. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs. R. Cai: Owner/Co-Owner; Self; R.C. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs. A.V. Schally: Owner/Co-Owner; Self; A.V.S. is co-inventor on GHRH agonists MR-409 patent, assigned to the University of Miami and the Veterans Affairs.. E. Bernal-Mizrachi: None. Patients with type 1 diabetes (T1D) have decreased functional β-cell mass due to inflammatory cell infiltration and cytokine-induced β-cell death. Clinical trials using a combination of immunomodulatory agents and β-cell protecting medications such as GLP1-R agonists (GLP1-Ra) are being considered. A potential limitation for the clinical efficacy of GLP1-R agonists is the loss of GLP-1-R at the cell surface in states of hyperglycemia. Hence, novel agents that enhance β-cell survival and proliferation through parallel pathways could improve responses to GLP1-R agonists and/or synergistically potentiate β-cell responses. Previous studies have demonstrated beneficial effects of growth hormone-releasing hormone receptor agonists (GHRH-Ra), such as MR-409 on islet preconditioning in transplantation models. Our previous studies showed that GHRH-Ra protects β-cells from autoimmune injury in vitro and improves glucose levels in a model of high-dose streptozotocin-induced diabetes via stimulation of the cAMP/PKA/CREB/IRS2 axis. However, the effects of combination of incretin-based therapy with GHRHRa on β-cell survival in T1D models have not yet been explored. Using the low-dose STZ model in C57/B6 mice, we investigated how MR-409 alone or in combination with GLP1-R agonists (Exendin-4 or Ex-4) promotes β-cell survival and prevents T1D. Low-dose STZ treated mice received daily MR-409 and Ex-4 injections, either alone or in combination for six weeks. We found that, STZ-treated mice that received either MR-409, Ex-4 or combination exhibited better glucose homeostasis while the combination failed to induce any additional effect. Interestingly, glucose tolerance and insulin levels were improved only in the MR-409-treated mice. β-cell mass was similarly increased in mice that received MR-409, Ex-4 and combination groups compared to the vehicle-treated. Further, treatment of human islets with MR-409 and/or Ex-4 induced insulin receptor substrate 2 (IRS2) expression, a master regulator of survival and growth in β-cells, while the combination of both therapies did not have any additional effect. Experiments in human islets exposed to proinflammatory cytokines treatment demonstrated that MR-409 and/or Ex-4 was associated with a decrease in β-cell death and improved insulin secretory function by activation of the cAMP/PKA/CREB/IRS2 axis. These studies demonstrate that although there was no evidence for a synergistic effect between MR-409 and Ex-4, MR-409 appears to have more potent effects on improving glucose tolerance and insulin levels when compared to the long-term effect of Ex-4 in a low-dose STZ model, suggesting that MR-409 could be an alternative therapeutic agent in the treatment of β-cell death in T1D. Presentation: 6/2/2024

Investigating the impact of a novel GHRHR gene variant on growth traits in Damascus goats

This study investigates the genotype and allele frequencies of three novel SNP loci (c.4218 T>G, c.2203 T>C, and c.7966 A>C) in the GHRHR gene within the Damascus goat breed and their associations with growth traits. The genotype frequencies for c.4218 T>G were 0.65 (TT), 0.15 (TG), and 0.20 (GG), with allele frequencies of 0.73 (T) and 0.27 (G), showing a significant deviation from Hardy-Weinberg equilibrium (HWE). The c.2203 T>C locus had genotype frequencies of 0.75 (TT) and 0.25 (CC), and the c.7966 A>C locus had 0.81 (AA) and 0.19 (CC), both consistent with HWE. Association analysis revealed significant correlations between the c.4218 T>G SNP and body weight and rump width, with the TT genotype showing the highest averages for both traits. The c.2203 T>C SNP was significantly associated with body weight and chest width, with the TT genotype again showing higher averages. The c.7966 A>C SNP was associated with chest depth and body length, with the AA genotype displaying lower averages for these traits. In silico predictions using multiple computational tools indicated that the identified missense SNP (p.31Ile>Ser) is novel and likely deleterious to the GHRHR protein's function and stability. Molecular docking demonstrated that the wild-type GHRHR binds more effectively with G protein alpha compared to the mutated form, predicting a mechanism through which this variant reduces the interactions of GHRHR with its cognate proteins. These findings provide valuable insights into the genetic factors influencing growth traits in Damascus goats and highlight the potential impact of the p.31Ile>Ser SNP on GHRHR functionality. This research underscores the importance of incorporating genetic markers in breeding programs to enhance growth traits and overall productivity.

Identification of Small-Molecule Antagonists Targeting the Growth Hormone Releasing Hormone Receptor (GHRHR).

The growth hormone-releasing hormone receptor (GHRHR) belongs to Class B1 of G protein-coupled receptors (GPCRs). Class B1 GPCR peptides such, as growth hormone-releasing hormone (GHRH), have been proposed to bind in a two-step model, where first the C-terminal region of the peptide interacts with the extracellular domain of the receptor and, subsequently, the N-terminus interacts with the seven transmembrane domain of the receptor, resulting in activation. The GHRHR has recently been highlighted as a promising drug target toward several types of cancer and has been shown to be overexpressed in prostate, breast, pancreatic, and ovarian cancer. Indeed, peptide GHRHR antagonists have displayed promising results in many cancer models. However, no nonpeptide GHRHR-targeting compounds have yet been identified. We have utilized several computational tools to target GHRHR and identify potential small-molecule compounds directed at this receptor. These compounds were validated in vitro using a cyclic adenosine monophosphate (cAMP) ELISA to measure activity at the GHRHR. In vitro results suggest that several of the novel small-molecule compounds could inhibit GHRH-induced cAMP accumulation. Preliminary analysis of the specificity/selectivity of one of the most effective hit compounds indicated that the effect seen was via inhibition of the GHRHR. We therefore report the first nonpeptide antagonists of GHRHR and propose a structural basis for inhibition induced by the compounds, which may assist in the future design of lead GHRHR compounds for treating disorders attributed to dysregulated/aberrant GHRHR signaling.

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in a Cohort of Hungarian Pediatric Patients with Hematological and Oncological Disorders: A Pilot Study.

Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.

Whole transcriptome analysis to identify non-coding RNA regulators and hub genes in sperm of non-obstructive azoospermia by microarray, single-cell RNA sequencing, weighted gene co-expression network analysis, and mRNA-miRNA-lncRNA interaction analysis

BackgroundThe issue of male fertility is becoming increasingly common due to genetic differences inherited over generations. Gene expression and evaluation of non-coding RNA (ncRNA), crucial for sperm development, are significant factors. This gene expression can affect sperm motility and, consequently, fertility. Understanding the intricate protein interactions that play essential roles in sperm differentiation and development is vital. This knowledge could lead to more effective treatments and interventions for male infertility.Materials and methodsOur research aim to identify new and key genes and ncRNA involved in non-obstructive azoospermia (NOA), improving genetic diagnosis and offering more accurate estimates for successful sperm extraction based on an individual’s genotype.ResultsWe analyzed the transcript of three NOA patients who tested negative for genetic sperm issues, employing comprehensive genome-wide analysis of approximately 50,000 transcript sequences using microarray technology. This compared gene expression profiles between NOA sperm and normal sperm. We found significant gene expression differences: 150 genes were up-regulated, and 78 genes were down-regulated, along with 24 ncRNAs up-regulated and 13 ncRNAs down-regulated compared to normal conditions. By cross-referencing our results with a single-cell genomics database, we identified overexpressed biological process terms in differentially expressed genes, such as “protein localization to endosomes” and “xenobiotic transport.” Overrepresented molecular function terms in up-regulated genes included “voltage-gated calcium channel activity,” “growth hormone-releasing hormone receptor activity,” and “sialic acid transmembrane transporter activity.” Analysis revealed nine hub genes associated with NOA sperm: RPL34, CYB5B, GOL6A6, LSM1, ARL4A, DHX57, STARD9, HSP90B1, and VPS36.ConclusionsThese genes and their interacting proteins may play a role in the pathophysiology of germ cell abnormalities and infertility.

Functional characterization of growth hormone releasing hormone and its receptor in amphioxus with implication for origin of hypothalamic-pituitary axis

Growth hormone-releasing hormone (GHRH) has been widely shown to stimulate growth hormone (GH) production via binding to GHRH receptor GHRHR in various species of vertebrates, but information regarding the functional roles of GHRH and GHRHR in the protochordate amphioxus remains rather scarce. We showed here that two mature peptides, BjGHRH-1 and BjGHRH-2, encoded by BjGHRH precursor, and a single BjGHRHR protein were identified in the amphioxus Branchiostoma. japonicum. Like the distribution profiles of vertebrate GHRHs and GHRHRs, both the genes Bjghrh and Bjghrhr were widely expressed in the different tissues of amphioxus, including in the cerebral vesicle, Hatschek’s pit, neural tube, gill, hepatic caecum, notochord, testis and ovary. Moreover, both BjGHRH-1 and BjGHRH-2 interacted with BjGHRHR, and triggered the cAMP/PKA signal pathway in a dose-dependent manner. Importantly, BjGHRH-1 and BjGHRH-2 were both able to activate the expression of GH-like gene in the cells of Hatschek’s pit. These indicate that a functional vertebrate-like GHRH-GHRHR axis had already emerged in amphioxus, which is a seminal innovation making physiological divergence including reproduction, growth, metabolism, stress and osmoregulation possible during the early evolution of vertebrates.

Effects of MIA-602, GHRH receptor antagonist, on emotional disorders in mice

Effects of MIA-602, GHRH receptor antagonist, on emotional disorders in mice

Skin assessment in congenital untreated isolated GH deficiency.

The separation between the inside and outside through the skin was fundamental for the evolution of prevertebrates, which grow through extrapituitary circuits, to vertebrates, which grow through the somatotrophic axis, namely pituitary growth hormone (GH). and circulating IGF1.Individuals with untreated isolated growth hormone (GH) deficiency (IGHD) due to a mutation in the GH-releasing hormone receptor (GHRH) gene, residing in Itabaianinha, Brazil, are vulnerable to skin cancer and have reduced sweating. However other aspects of their skin physiology are still unknown. Our objectives were to evaluate the number of skin cancers, skin aging, and functional aspects of the skin in this IGHD cohort. Twenty-six IGHD individuals and 26 controls matched by age, sex, ethnicity, and occupation were submitted to a biochemical, dermatological and a functional skin assessment by the Multi Probe Adapter Cutometer® MPA 580. There was no difference in the number of skin cancers and in the degrees of photodamage between the groups. The melanin content in the forearm was similar between the groups but was lower in the buttocks (p = 0.005), as well as skin resistance (p < 0.0001) and elasticity (p = 0.003), lower in the IGHD. There was no difference in hydration and sebum content between the two groups. IGHD is apparently associated with a neutral profile in terms of skin cancer and photodamage, with similar melanin on the forearm and lower buttocks, lower skin resistance and elasticity, with hydration and sebum similar to controls.

The allostery and modification of hGHRH molecules and specific dimer produced significant fertility effect by proliferating and activating in-situ ovarian mesenchymal stem cells

The negative coordination of growth hormone secretagogue receptor (GHS-R) and growth hormone-releasing hormone receptor (GHRH-R) involves in the repair processes of cellular injury. The allosteric U- or H-like modified GHRH dimer Grinodin and 2Y were comparatively evaluated in normal Kunming mice and hamster infertility models induced by CPA treatment. 1–3–9 µg of Grinodin or 2Y per hamster stem-cell-exhaustion model was subcutaneously administered once a week, respectively inducing 75–69–46 or 45–13–50 % of birth rates. In comparison, the similar mole of human menopausal gonadotropin (hMG) or human growth hormone (hGH) was administered once a day but caused just 25 or 20 % of birth rates. Grinodin induced more big ovarian follicles and corpora lutea than 2Y, hMG, hGH. The hMG-treated group was observed many distorted interstitial cells and more connective tissues and the hGH-treated group had few ovarian follicles. 2Y had a plasma lifetime of 21 days and higher GH release in mice, inducing lower birth rate and stronger individual specificity in reproduction as well as only promoting the proliferation of mesenchymal-stem-cells (MSCs) in the models. In comparison, Grinodin had a plasma lifetime of 30 days and much lower GH release in mice. It significantly promoted the proliferation and activation of ovarian MSCs together with the development of follicles in the models by increasing Ki67 and GHS-R expressions, and decreasing GHRH-R expression in a dose-dependent manner. However, the high GH and excessive estrogen levels in the models showed a dose-dependent reduction in fertility. Therefore, unlike 2Y, the low dose of Grinodin specifically shows low GHS-R and high GHRH-R expressions thus evades GH and estrogen release and improves functions of organs, resulting in an increase of fertility.

Brain morphometry and estimation of aging brain in subjects with congenital untreated isolated GH deficiency.

Individuals with isolated GH deficiency (IGHD) due to a mutation in the GHRH receptor gene have a normal life expectancy and above 50years of age, similar total cognitive performance, with better attention and executive function than controls. Our objectives were to evaluate their brain morphometry and brain aging using MRI. Thirteen IGHD and 14 controls matched by age, sex, and education, were enrolled. Quantitative volumetric data and cortical thickness were obtained by automatic segmentation using Freesurfer software. The volume of each brain region was normalized by the intracranial volume. The difference between the predicted brain age estimated by MRI using a trained neuronal network, and the chronological age, was obtained. p < 0.005 was considered significant and 0.005 < p < 0.05 as a suggestive evidence of difference. In IGHD, most absolute values of cortical thickness and regional brain volumes were similar to controls, but normalized volumes were greater in the white matter in the frontal pole and in the insula bilaterally, and in the gray matter, in the right insula and in left Caudate (p < 0.005 for all comparisons) We also noticed suggestive evidence of a larger volume in IGHD in left thalamus (p = 0.006), right thalamus (p = 0.025), right caudate (p = 0.046) and right putamen (p = 0.013). Predicted brain ages were similar between groups. IGHD is primarily associated with similar absolute brain measurements, and a set of larger normalized volumes, and does not appear to alter the process of brain aging.

Exploring the role of GHRH antagonist MIA-602 in overcoming Doxorubicin-resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the rapid proliferation of mutagenic hematopoietic progenitors in the bone marrow. Conventional therapies include chemotherapy and bone marrow stem cell transplantation; however, they are often associated with poor prognosis. Notably, growth hormone-releasing hormone (GHRH) receptor antagonist MIA-602 has been shown to impede the growth of various human cancer cell lines, including AML. This investigation examined the impact of MIA-602 as monotherapy and in combination with Doxorubicin on three Doxorubicin-resistant AML cell lines, KG-1A, U-937, and K-562. The in vitro results revealed a significant reduction in cell viability for all treated wild-type cells. Doxorubicin-resistant clones were similarly susceptible to MIA-602 as the wild-type counterpart. Our in vivo experiment of xenografted nude mice with Doxorubicin-resistant K-562 revealed a reduction in tumor volume with MIA-602 treatment compared to control. Our study demonstrates that these three AML cell lines, and their Doxorubicin-resistant clones, are susceptible to GHRH antagonist MIA-602.

Abstract 4620: Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is characterized by the uncontrolled proliferation of immature hematopoietic progenitors, often associated with a poor prognosis. Conventional therapies involve chemotherapy and bone marrow stem cell transplantation. The growth hormone releasing hormone receptor (GHRH-R) antagonist MIA-602 has exhibited inhibitory effects on the growth of various human cancer cell lines, including AML cells. This study aimed to investigate the impact of MIA-602 alone and in combination with doxorubicin on three doxorubicin-resistant AML cell lines, K-562, U-937, and KG-1A. Initial examinations confirmed the presence of GHRH receptors in both wild-type and doxorubicin-resistant cell lines through western blot analysis. Doxorubicin was administered at concentrations of 0.005, 0.01, and 0.05 μg/ml, while MIA-602 was used at 5 μmol/L. Subsequent experiments involved culturing both wild-type and doxorubicin-resistant clones with MIA-602 at concentrations ranging from 0.05 μmol/L to 5 μmol/L for 24 and 48 hours. The results revealed a significant, dose- and time-dependent reduction in cell proliferation across all six cell lines. Both wild-type and doxorubicin-resistant clones exhibited a comparable decrease in cell proliferation when exposed to MIA-602 at concentrations greater than 0.05 μmol/L (p &amp;lt; 0.05) after 24 and 48 hours. In conclusion, our study demonstrates that these three AML cell lines and their doxorubicin-resistant clones remain susceptible to GHRH antagonist MIA-602. These findings may pave the way for the development of novel therapies or drug combinations using GHRH antagonists such as MIA-602 for treating AML. This is of particular interest for those who are resistant to conventional chemotherapy, further broadening the spectrum of treatment options available. Additional investigations are warranted to progress these findings to in vivo xenograft models. Citation Format: Simonetta I. Gaumond, Joel Costoya, Andrew V. Schally, Joaquin J. Jimenez. Exploring the role of GHRH antagonist MIA-602 in overcoming doxorubicin resistance in acute myeloid leukemia [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 4620.

Growth Hormone-Releasing Hormone as Potential Diagnostic and Therapeutic Target in Colorectal Cancer

Abstract: As the third most prevalent cancer, there is no convincing treatment for colorectal cancer. Growth Hormone-releasing Hormone (GHRH) is an important hormone for the growth and maturity of humans and other animals. Also, recent studies show both GHRH and GHRH receptor to be expressed in many types of cancers, such as lung, breast, colon, ovarian, gastric, and pancreatic cancer. GHRH has been shown to augment the growth and proliferation of cancer cells in in vivo and in vitro studies, but studies have shown that its antagonist can inhibit the growth or even metastasis of cancer cells. The development of many classes of GHRH-R antagonists over the past three decades has shown strong in vivo and in vitro growth inhibitory effects on cancer. The present study is an attempt to review the studies on the association between GHRH and colorectal cancer and examine if the hormone can be targeted for therapy or used as a prognostic marker for colorectal cancer or not.

Identification and functional analysis of first heterozygous frameshift mutation in the GHRH gene in a Chinese boy with isolated growth hormone deficiency

BackgroundIsolated growth hormone deficiency (IGHD) is a rare genetically heterogeneous disorder caused primarily by mutations in GH1 and GH releasing hormone receptor (GHRHR). The aim of this study was to identify the molecular etiology of a Chinese boy with IGHD. MethodsWhole-exome sequencing, sanger sequencing and bioinformatic analysis were performed to screen for candidate mutations. The impacts of candidate mutation on gene expression, intracellular localization and protein function were further evaluated by in vitro assays. ResultsA novel heterozygous frameshift mutation in the GHRH gene (c.91dupC, p.R31Pfs*98) was identified in a Chinese boy clinically diagnosed as having IGHD. The mutation was absent in multiple public databases, and considered as deleterious using in silico prediction, conservative analysis and three-dimensional homology modeling. Furthermore, mRNA and protein expression levels of mutant GHRH were significantly increased than wild-type GHRH (p < 0.05). Moreover, mutant GHRH showed an aberrant accumulation within the cytoplasm, and obviously reduced ability to stimulate GH secretion and cAMP accumulation in human GHRHR-expressing pituitary GH3 cells compared to wild-type GHRH (p < 0.05). ConclusionOur study discovered the first loss-of function mutation of GHRH in a Chinese boy with IGHD and provided new insights on IGHD pathogenesis caused by GHRH haploinsufficiency.

Screening of GHSR, GHRHR, GH1 genes in isolated growth hormone deficiency disease in Egyptian patients

Abstract Background Isolated growth hormone deficiency (IGHD) is a hereditary disorder that causes significant short stature. GHD has a reported incidence of 1/4000–1/10,000 births. It is caused by mutations in the major somatotroph axis genes, involving GH1, codes for growth hormone, GHSR, and GHRHR, codes for growth hormone secretagogue receptor and growth hormone-releasing hormone receptor, respectively. Aims of the study The present study aims to examine the clinical phenotype and investigate the genetic etiology of ten Egyptian patients with type I isolated growth hormone insufficiency. Patients and methods Patients recruited for the study were clinically diagnosed by two provocation tests and were subjected to a thorough history, clinical examination, and anthropometric measurements. Sanger sequencing and mutational analysis of the three genes, GH1, GHSR, and GHRHR, was our approach, performed in all enrolled IGHD patients. The variants identified were analyzed using the biological, population, sequence variants, and clinical genetics databases. Prediction of the pathogenicity of the novel variants was done by in silico prediction tools following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results Sanger sequencing revealed a previously reported pathogenic mutation (NM_000823.4: c.1069C &gt; T; p.Arg357Cys) in the GHRHR gene in one patient and a novel frameshift variant (NM_198407.2: c.1043dup; Ser349Leu fs*6) in the GHSR gene in another patient. This is the fourth report highlighting the autosomal dominant inheritance of the GHSR mutation as a cause of isolated growth hormone deficiency. A number of previously reported variants, but of rare frequency, were identified in this study. In our IGHD cases, 90% of the patients were underweight, 50% had anemia, and 80% showed hypovitaminosis D. Conclusion Our findings broaden the mutational spectrum underlying the IGHD in Egyptian patients and point out the importance of mutation screening of the GHSR and GHRHR genes. This study also acknowledges the autosomal dominant mode of inheritance of the GHSR mutation as a cause for dwarfism.

Cognitive performance during senescence in untreated congenital isolated GH deficiency.

Individuals with untreated isolated GH deficiency (IGHD) due to a mutation in the GHRH receptor gene from Itabaianinha Brazil have increased insulin sensitivity, normal life expectancy, and an extended health span, i.e. the period of life free from disabilities. We hypothesize that their prolonged health span is accompanied by a delayed cognitive decline in senescence. To test this hypothesis, we have administered the Literacy-Independent Cognitive Assessment (LICA) to 15 IGHD individuals aged over 50 years and 15 controls matched by age, sex, years of education, and percentage of illiteracy. All individuals were negative for HIV and syphilis serology, and there were no differences in serum levels of folate, vitamin B12 and TSH between the two groups, while free T4 was higher in the IGHD group. IGHD subjects had a higher total LICA score than controls, 215 (22.7) vs 204.2 (18.1), without reaching statistical significance. Scores of memory, visuoconstruction, language and calculation were similar between the two groups, with better attention (9.5 (1.4) vs 8.3 (1.1), P = 0.01) and executive function (38.3 (4.8) vs 35.1 (2.5), P = 0.03) scores in IGHD. MANCOVA revealed that group (but no age) had a significant effect on the LICA variables (partial eta squared of 0.455, power of 0.812, P = 0.02). This effect is verified on attention (partial eta squared 0.216, power of 0.749, P = 0.01) and executive function (partial eta squared 0.154, power of 0.570, P = 0.03. In conclusion, IGHD in senescence is associated with similar total cognitive performance but better attention and executive function than controls.