Notch signaling as a novel target for uterine leiomyosarcoma (259)

Abstract

<b>Objectives:</b> The Notch signaling pathway is evolutionarily conserved and has been implicated as a potential therapeutic target in several cancers. Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with poor survival, higher prevalence in Black women than White women, and few effective adjuvant therapies. The objective of this study was to determine the expression of Notch in uLMS as a foundation for investigating the Notch signaling pathway as a potential therapeutic target in uLMS. <b>Methods:</b> We analyzed previously published mRNA sequencing (seq) datasets on LMS, including uLMS and other sarcoma patient tissue samples for expression of Notch pathway genes. We performed immunostaining for <i>NOTCH1</i> and <i>NOTCH3</i> on uterine tissue from benign hysterectomies (BH, <i>n</i>=3) and uLMS samples (<i>n</i>=30) from a multiple organ LMS tissue array (US Biomax, Inc). <i>NOTCH1</i> expression was measured as a percent positive stain per area, and <i>NOTCH3</i> expression was quantified with an H-Score. We analyzed Notch intracellular domain (ICD) protein expression in human uterine smooth muscle cell lines (hUT-SMC) and uLMS cell lines, SK-UT-1B and SK-LMS-1, by Western blot, and expression of Notch effectors, <i>HES1, HEY1,</i> and <i>NRARP,</i> by qPCR and immunofluorescence. Numerical data were reported as mean ± SEM or median ± interquartile range and compared using unpaired t-test or Mann-Whitney U, with p≤0.05 being statistically significant. <b>Results:</b> mRNA seq analysis revealed similar levels of expression for <i>NOTCH1, 2,</i> and <i>3</i> and <i>HES1</i> in LMS compared to other sarcomas. <i>NOTCH4</i> was not included in the original mRNA seq data and thus could not be evaluated for expression levels. Immunostaining revealed that median H-score of <i>NOTCH3</i> in uLMS was higher than in BH (211.4, IQR: 165.6-239.3 vs 100.3, IQR: 100.0-100.5, p≤0.05), whereas mean expression of <i>NOTCH1</i> was similar (3.7 ± 1.6% vs 1.9 ± 0.5%, p>0.05). Western blot analysis demonstrated that ICD for <i>NOTCH1-4</i> was expressed in hUT-SMC, N1ICD and N2ICD were expressed in SK-UT-1B, and N1ICD, N2ICD, and N4ICD were expressed in SK-LMS-1. mRNA expression of <i>HES1, HEY1,</i> and <i>NRARP</i> and nuclear expression of <i>HES1</i> indicate active Notch signaling in SK-LMS-1 and SK-UT-1B cells. <b>Conclusions:</b> Notch is expressed in uLMS patient tissues and uLMS cell lines. Notch signaling and distinct expression of Notch proteins in uLMS cell lines support the investigation of Notch inhibition as a potential target for treatment options in uLMS. Further studies are warranted to determine the efficacy of targeting Notch inhibition using therapies, such as gamma-secretase inhibitors alone or in combination with a cytotoxic agent.