Hematological and Neurological Expressed 1 (HN1): a predictive and pharmacodynamic biomarker of metformin response in endometrial cancers

Preoperative use of metformin in obese women with endometrioid endometrial cancer (EEC) reduces tumor proliferation and inhibits the mammalian target of rapamycin (mTOR) pathway, though is only effective in select cases. This study sought to identify a predictive and/or pharmacodynamic proteomic signature of metformin response to tailor its pharmacologic use. Matched pre- and post-metformin treated tumor tissues from a recently completed phase 0 window trial of metformin in EEC patients (ClinicalTrials.gov: [NCT01911247][1]), were analyzed by mass spectrometry (MS)-based proteomic and immunohistochemical analyses. Hematological and neurological expressed 1 (HN1) was significantly elevated in metformin responders (n=13) versus non-responders (n=7), which was also found to decrease in abundance in metformin responders following treatment; observations that were verified by immunohistochemical staining for HN1. Metformin response and loss of HN1 was assessed in RL95-2 and ACI-181 endometrial cancer cell lines. We further identified that silencing of HN1 abundance does not alter cellular response to metformin or basal cell proliferation, but that HN1 abundance does decrease in response to metformin treatment in RL95-2 and ACI-181 endometrial cancer cell lines. These data suggest that HN1 represents a predictive and pharmacodynamic biomarker of metformin response that, if validated in larger patient populations, may enable pre-operative EEC patient stratification to metformin treatment and the ability to monitor patient response. Novelty and Impact Hematological and Neurological Expressed 1 (HN1) is elevated in endometrioid endometrial cancer (EEC) patients that respond to metformin vs non-responders and decreases after treatment. HN1 decreases after metformin treatment of EEC cells in vitro, but is not necessary for metformin response or proliferation. We report HN1 as a novel predictive and pharmacodynamic biomarker of metformin response in EEC that may enable pre-operative EEC patient stratification to metformin treatment and the ability to monitor patient response. * HN1 : Hematological and Neurological Expressed 1 JPT1 : Jupiter microtubule associated homolog 1 EC : endometrial cancer EEC : cndometrioid endometrial cancer Ki-67 : Antigen Ki-67 MS : mass spectrometry MS/MS : tandem mass spectrometry LC : liquid chromatography mTOR : mammalian target of rapamycin AMPK : adenosine monophosphate kinase ATP : adenosine triphosphate LMD : laser microdissection H&E : hematoxylin and eosin IHC : Immunohistochemistry PSMs : peptide spectral matches FDR : false discovery rate TMA : tissue microarray CO2 : carbon dioxide siRNA : small interfering RNA mM : millimolar MTS : 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium siNT : small interfering non-targeting RNA siHN1 : small interfering HN1 RNA qPCR : quantitative polymerase chain reaction cDNA : complimentary DNA DNA : deoxyribonucleic acid RNA : ribonucleic acid GAPDH : glyceraldehyde 3-phosphate dehydrogenase SDS : sodium dodecyl sulfate NaCl : sodium chloride PVDF : polyvinylidene difluoride AKT : protein kinase B IgG : immunoglobulin HRP : horseradish peroxidase IP-MS : immunoprecipitation mass spectrometry EDTA : ethylenediaminetetraacetic acid TCGA : The Cancer Genome Atlas RNA-seq : RNA sequencing LD50 : lethal dose 50 mRNA : messenger RNA MYC : MYC oncogene Mg : milligram logFC : Log fold-change CTNNB1 : beta catenin S473 : serine 473 PCNA : proliferating cell nuclear antigen p-S6 kinase : phosphorylated ribosomal protein S6 kinase beta-1 p-EIF4-BP1 : phosphorylated Eukaryotic translation initiation factor 4E-binding protein 1 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01911247&atom=%2Fbiorxiv%2Fearly%2F2019%2F04%2F09%2F602870.atom