High Throughput Transcriptome Data Analysis and Computational Verification Reveal Immunotherapy Biomarkers of Compound Kushen Injection for Treating Triple-Negative Breast Cancer.

Xinkui Liu,Peizhi Ye,Shan Lu,Zhihong Huang,Dechao Bu,Yingying Zhang,Haojia Wang,Yurong Song,Chao Wu,Jiarui Wu,Liangliang Shen,Yang Wu,Jingyuan Zhang,Changgeng Fu,Fengying Guo,Xianchun Duan,Yi Zhao

doi:10.3389/fonc.2021.747300

Abstract

BackgroundAlthough notable therapeutic and prognostic benefits of compound kushen injection (CKI) have been found when it was used alone or in combination with chemotherapy or radiotherapy for triple-negative breast cancer (TNBC) treatment, the effects of CKI on TNBC microenvironment remain largely unclear. This study aims to construct and validate a predictive immunotherapy signature of CKI on TNBC.MethodsThe UPLC-Q-TOF-MS technology was firstly used to investigate major constituents of CKI. RNA sequencing data of CKI-perturbed TNBC cells were analyzed to detect differential expression genes (DEGs), and the GSVA algorithm was applied to explore significantly changed pathways regulated by CKI. Additionally, the ssGSEA algorithm was used to quantify immune cell abundance in TNBC patients, and these patients were classified into distinct immune infiltration subgroups by unsupervised clustering. Then, prognosis-related genes were screened from DEGs among these subgroups and were further overlapped with the DEGs regulated by CKI. Finally, a predictive immunotherapy signature of CKI on TNBC was constructed based on the LASSO regression algorithm to predict mortality risks of TNBC patients, and the signature was also validated in another TNBC cohort.ResultsTwenty-three chemical components in CKI were identified by UPLC-Q-TOF-MS analysis. A total of 3692 DEGs were detected in CKI-treated versus control groups, and CKI significantly activated biological processes associated with activation of T, natural killer and natural killer T cells. Three immune cell infiltration subgroups with 1593 DEGs were identified in TNBC patients. Then, two genes that can be down-regulated by CKI with hazard ratio (HR) > 1 and 26 genes that can be up-regulated by CKI with HR < 1 were selected as key immune- and prognosis-related genes regulated by CKI. Lastly, a five-gene prognostic signature comprising two risky genes (MARVELD2 and DYNC2I2) that can be down-regulated by CKI and three protective genes (RASSF2, FERMT3 and RASSF5) that can be up-regulated by CKI was developed, and it showed a good performance in both training and test sets.ConclusionsThis study proposes a predictive immunotherapy signature of CKI on TNBC, which would provide more evidence for survival prediction and treatment guidance in TNBC as well as a paradigm for exploring immunotherapy biomarkers in compound medicines.

Highlights

Triple-negative breast cancer (TNBC) is the most malignant and aggressive subtype of breast cancer, which is pathologically featured by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression [1,2,3]
A recent study aimed at illustrating the effect of Compound kushen injection (CKI) on tumor immunity demonstrates that CKI relieves the immunosuppression mediated by tumorassociated macrophages and afterwards alleviates the immunosuppressive effects on CD8+ T cells, which enhances the efficacy of low-dose sorafenib and avoids chemotherapyinduced adverse effects [10]
We identified a total of 23 chemical components from CKI by using UPLC-Q-TOF-MS analysis (Figure 1 and Table 1)

Summary

Introduction

Triple-negative breast cancer (TNBC) is the most malignant and aggressive subtype of breast cancer, which is pathologically featured by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression [1,2,3]. CKI has been widely used alone or in combination with chemotherapy or Abbreviations: CKI, compound kushen injection; TNBC, triple-negative breast cancer; TME, tumor microenvironment; UPLC-Q-TOF-MS, ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry; RNA-seq, RNA sequencing; PCA, principal component analysis; DEGs, differential expression genes; GSVA, gene set variation analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DSS, disease specific survival; OS, overall survival; MFS, metastasis-free survival; ssGSEA, single sample gene set enrichment analysis; NES, normalized enrichment score; LASSO, least absolute shrinkage and selection operator; DCs, dendritic cells; NK cells, natural killer cells; MDSCs, myeloid-derived suppressor cells; NKT cells, natural killer T cells; pDCs, plasmacytoid dendritic cells; Tcm cells, central memory T cells; Tem cells, effector memory T cells; gd T cells, gamma delta T cells; Treg cells, regulatory T cells; Tfh cells, T follicular helper cells; Th1 cells, type 1 T helper cells; Th2 cells, type 2 T helper cells; Th17 cells, type 17 T helper cells. Notable therapeutic and prognostic benefits of compound kushen injection (CKI) have been found when it was used alone or in combination with chemotherapy or radiotherapy for triple-negative breast cancer (TNBC) treatment, the effects of CKI on TNBC microenvironment remain largely unclear. This study aims to construct and validate a predictive immunotherapy signature of CKI on TNBC

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Conclusion