IntroductionInsulin-like peptide 5 (INSL5) is a peptide hormone with proposed actions in glucose homeostasis and appetite regulation via its cognate receptor, relaxin family peptide receptor 4 (RXFP4). Here, we look for evidence for their involvement in the immune system using a mouse model.MethodsIn silico analyses: we queried public databases for evidence of expression of INSL5-RXFP4 in immune system tissues/cells (NCBI’s SRA and GeoProfiles) and disorders (EMBO-EBI) and performed phylogenetic footprinting to look for evidence that they are regulated by immune-associated transcription factors (TFs). Experimental analyses: We characterized the expression and correlation of INSL5/RXFP4 and other immune system markers in central and peripheral immune organs from C57/bl6 mice in seven cohorts. We tested whether fluctuations in circulating INSL5 induce an immune response, by injecting mice with 30 μg/kg of INSL5 peptide in the peritoneum, and examining levels of immune markers and metabolic peptides in plasma. Lastly, we quantified the expression of Rxfp4 in T-cells, dendritic cells and cell lines derived from human and mouse and tested the hypothesis that co-incubation of ANA-1 cells in INSL5 and LPS alters cytokine expression.ResultsWe find Insl5 expression only in thymus (in addition to colon) where its expression was highly correlated with Il-7, a marker of thymocyte development. This result is consistent with our in silico findings that Insl5 is highly expressed in thymic DP, DN thymocytes and cortical TEC’s, and with evidence that it is regulated by thymocyte-associated TF’s. We find Rxfp4 expression in all immune organs, and moderately high levels in DCs, particularly splenic DCs, and evidence that it is regulated by immune-associated TF’s, such as STAT’s and GATA. Systemic effects: We observed significantly elevated concentrations of blood GLP-1, GIP, GCG and PYY following intraperitoneal injection of INSL5, and significantly altered expression of cytokines IL-5, IL-7, M-CSF, IL-15, IL-27 and MIP-2. Immune cell effects: Incubation of ANA-1 cells with INSL5 impeded cell growth and led to a transient elevation of IL-15 and sustained reduction in IL-1β, IL-6 and TNFα.ConclusionWe propose that INSL5-RXFP4 play a novel role in both central and peripheral immune cell signaling.

Background: Breast cancer (BC) is the most diagnosed cancer worldwide and the most common cancer diagnosed in American women. Despite successes of adjuvant endocrine therapies in treating the most common subtype of hormone receptor-positive (HR+) BC, de novo and emergent resistance to these therapies, i.e., endocrine resistance, remains a significant concern with patients experiencing recurrences during and after adjuvant treatment. We report here unique patterns of expression (UPE) of N-myristoyltransferase 1 (NMT1) protein that are prognostic and predictive markers for HR+ BC. We have discovered that the expression and localization of NMT1 are predictive markers for the endocrine therapy response in HR+ BC. The data from many observational studies suggest that after five years of adjuvant endocrine treatment, the likelihood of distant recurrence continues for the subsequent twenty years. We have developed simple immunohistochemical (IHC)-based tests that could become part of routine first line prognostic and predictive tests and have the potential to be incorporated as necessary tests in the panel of BC tests for designing treatment regimens of BC. Methods: Expression patterns of NMT1 were determined using IHC analyses on formalin fixed paraffin embedded primary tumor samples from treatment naïve BC patients. The tumor tissues in duplicates were stained with NMT1 monoclonal/polyclonal antibodies on an autostainer (Leica Bond). Stained slides were scored in a blinded manner to outcomes and scored with an "H" index representing the intensity and percent positive cells within each section. The relationship of UPE was correlated with clinical outcomes of RFS (RFS = endpoint recurrence and/or death due to BC) and OS (OS = endpoint death due to breast cancer). Results: The final cohort was composed of 448 BC cases of primary HR+ tumors from patients who received adjuvant tamoxifen therapy after surgery. UPE2 (defined by > median H score 100) was significantly associated with better clinical outcomes represented by both RFS (HR = 0.70, P = 0.0304, 95% CI 0.510 to 0.97, n = 440) and OS (HR = 0.71, P = 0.0306, 95% CI 0.530 to 0.97, n = 440), whereas UPE4 strongly predicted worse treatment response for both RFS (HR= 1.49, P = 0.0014, 95% CI 1.08 to 2.04, n=440) and OS (HR = 1.54, P = 0.0055, 95% CI = 1.54 to 2.08, n=440). Conclusions: We observed that the expression and localization patterns of NMT1 constitute four unique patterns of expression signatures (UPE:1-4) and serve as prognostic and potentially predictive markers for HR+ BC. This assay provides a novel method to stratify cancers that may better enable endocrine treatment and duration optimization in the adjuvant setting for HR+ breast cancer. Moreover, in distinction to genomic assays, fewer logistic and financial barriers to implementation would exist for the UPE:1-4 IHC assay in resource-poor settings. Citation Format: Danira Jaksic, Amandeep Kaur, Dean Reddick, David David Datzkiw, Shailly Varma Shrivastav, Vijayakrishna Gadi, Leigh Murphy, Anuraag Shrivastav. Novel immunohistochemistry-based predictive and prognostic tests for hormone receptor-positive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5460.

N-myristoylation refers to the attachment of a 14-carbon fatty acid onto the N-terminal glycine residue of a target protein. The myristoylation reaction, catalyzed by N-myristoyltrasnferase (NMT), is essential for regulating cellular activities such as signal transduction, proliferation, migration, differentiation, and transformation. Although a considerable amount of research is performed on the overexpression of NMT in pathogenic conditions, a fundamental knowledge gap exists on the evolution of NMT and the functional impact of myristoylation for normal cellular development and functions. We performed evolutionary analyses of the NMT gene and found that most non-vertebrates harbor a single nmt gene and all vertebrates examined harbor two genes; nmt1 and nmt2. For the first time, we report that teleosts harbor two copies of nmt1, named nmt1a and nmt1b. We traced the evolutionary history of the chromosomal fragments hosting NMT1 and NMT2 in humans and found that NMT1 and NMT2 trace back to a single vertebrate ancestral chromosome. We also report the presence of putative nuclear localization sequence (NLS) and amino acid residues flanking NLS. The presence of phosphorylatable amino acid residues flanking the NLS suggests that nuclear localization of NMT is regulated by phosphorylation. The nuclear localization of NMT suggest its potential role in gene transcription.