Serine Protease Inhibitor Kazal Type 1, A Potential Biomarker for the Early Detection, Targeting, and Prediction of Response to Immune Checkpoint Blockade Therapies in Hepatocellular Carcinoma

Abstract

BackgroundWe aimed to characterize serine protease inhibitor Kazal type 1 (SPINK1) as a gene signature for the early diagnosis, molecular targeting, and prediction of immune checkpoint blockade (ICB) treatment response of hepatocellular carcinoma (HCC).MethodsThe transcriptomics, proteomics, and phenotypic analyses were performed separately or in combination.ResultsWe obtained the following findings on SPINK1. Firstly, in the transcriptomic training dataset, which included 279 stage I and II tumor samples (out of 1,884 stage I–IV HCC specimens) and 259 normal samples, significantly higher area under curve (AUC) values and increased integrated discrimination improvement (IDI) and net reclassification improvement (NRI) were demonstrated for HCC discrimination in SPINK1-associated models compared with those of alpha-fetoprotein (AFP). The calibration of both SPINK1-related curves fitted significantly better than that of AFP. In the two independent transcriptomic validation datasets, which included 201, 103 stage I-II tumor and 192, 169 paired non-tumor specimens, respectively, the obtained results were consistent with the above-described findings. In the proteomic training dataset, which included 98 stage I and II tumor and 165 normal tissue samples, the analyses also revealed better AUCs and increased IDI and NRI in the aforementioned SPINK1-associated settings. A moderate calibration was shown for both SPINK1-associated models relative to the poor results of AFP. Secondly, in the in vitro and/or in vivo murine models, the wet-lab experiments demonstrated that SPINK1 promoted the proliferation, clonal formation, migration, chemoresistance, anti-apoptosis, tumorigenesis, and metastasis of HCC cells, while the anti-SPINK1 antibody inhibited the growth of the cells, suggesting that SPINK1 has “tumor marker” and “targetable” characteristics in the management of HCC. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that SPINK1 was engaged in immunity-related pathways, including T-cell activation. Thirdly, in the transcriptomic analyses of the 368 HCC specimens from The Cancer Genome Atlas (TCGA) cohort, the high abundance of SPINK1 was positively correlated with the high levels of activated tumor-infiltrating CD4+ and CD8+ T lymphocytes and dendritic and natural killer cells, while there were also positive correlations between SPINK1 and immune checkpoints, including PD-1, LAG-3, TIM-3, TIGIT, HAVCR2, and CTLA-4. The ESTIMATE algorithm calculated positive correlations between SPINK1 and the immune and ESTIMATE scores, suggesting a close correlation between SPINK1 and the immunogenic microenvironment within HCC tissues, which may possibly help in predicting the response of patients to ICB therapy.ConclusionsSPINK1 could be a potential biomarker for the early detection, targeted therapy, and prediction of ICB treatment response in the management of HCC.

SPINK1 Overexpression Correlates with Hepatocellular Carcinoma Treatment Resistance Revealed by Single Cell RNA-Sequencing and Spatial Transcriptomics

“…SPINK1 is an oncogenic protein that is overexpressed in various tumors, including HCC [ 6 , 7 ]. Previous investigations into SPINK1 have primarily focused on bulk tumors [ 16 , 34 ]. However, due to the significant inter-tumoral and intra-tumoral heterogeneity of HCC, a detailed exploration of SPINK1 at single cell level is urgently needed.…”

“…The remaining sub-clusters were consolidated as SPINK1-low HCC cells. In cohort 4, tumor sub-clusters numbered 2,5,6,7,8,12,14,15,17,18,19,21,22,24,27,30,32,34, and 35 demonstrated high SPINK1 expression, promoting the integration of these sub-clusters as SPINK1-high HCC cells. The remaining sub-clusters were integrated as SPINK1-low HCC cells.…”

SPINK1 is a Potential Diagnostic and Prognostic Biomarker for Sepsis

“…The literature review revealed that SPINK1 is abnormally expressed in various tumors, such as liver, breast, ovarian, lung, pancreatic, and renal cancers, and correlates with their overall survival rates. 20 SPINK1 is upregulated in several tumors at the transcriptome level, such as hepatocellular carcinoma 21 and prostate cancer. 22 However, the previous transcriptomic sequencing results of sepsis patients and healthy controls by our group did not reveal SPINK1 expression at the transcriptome level.…”

SPINK1-induced tumor plasticity provides a therapeutic window for chemotherapy in hepatocellular carcinoma

“…It will be interesting to also further explore how the suppression of SPINK1 affects the tumor microenvironment (TME), including immune cells; as well as what other cell types in the niche may secrete SPINK1 to promote HCC. In this connection, Lu et al previously found inflammatory SPINK1 to prevent cytolytic granule granzyme A-mediated apoptosis/immune-killing 47 ; and that SPINK1 secreted by stromal cells in a damaged tumor microenvironment following chemotherapy, can promote more aggressive cancer phenotypes 32 ; additionally, Jia et al demonstrated SPINK1 as a potential biomarker for the early detection, targeted therapy, and prediction of immune checkpoint blockade (ICB) treatment response in HCC patients 48 . By analyzing a scRNA-seq of a publicly available dataset (GSE151530) 49 , SPINK1 is found to be primarily expressed in malignant cells, with minimal expression in immune cells and stromal cells.…”