SPINK4 promotes colorectal cancer cell proliferation and inhibits ferroptosis.

BACKGROUND: Little is known about the role of serine peptidase inhibitor Kazal type 4 (SPINK4) in colorectal cancer (CRC) and ferroptosis. Therefore, this study aimed to determine the effect of SPINK4 on CRC pathogenesis and ferroptosis. METHODS: SPINK4 expression was analyzed in public datasets and examined using immunohistochemistry. The biological function of SPINK4 in CRC cell lines and its effect on ferroptosis were tested. An immunofluorescence assay was performed to determine the location of SPINK4 in cells, and mouse models were established to determine the effects of SPINK4 in vivo. RESULTS: CRC datasets and clinical samples analysis revealed that SPINK4 mRNA and protein levels were significantly reduced in CRC tissues compared to control tissues (P < 0.05). Two CRC cell lines (HCT116 and LoVo) were selected, and the in vitro and in vivo experiments showed that overexpression of SPINK4 greatly promotes the proliferation and metastasis of CRC cells and tumor growth (P < 0.05). The immunofluorescence assay indicated that SPINK4 is mainly located in the nucleoplasm and nucleus of CRC cells. Furthermore, SPINK4 expression was reduced after cell ferroptosis induced by Erastin, and overexpression of SPINK4 greatly inhibited ferroptosis in CRC cells. The results of mouse model further demonstrated that SPINK4 overexpression inhibited CRC cell ferroptosis and facilitated tumor growth. CONCLUSIONS: SPINK4 was decreased in CRC tissues and promoted cell proliferation and metastasis; overexpression of SPINK4 inhibited CRC cell ferroptosis.

Single-cell profiling reveals differences between human classical adenocarcinoma and mucinous adenocarcinoma.

Colorectal cancer is a highly heterogeneous disease. Most colorectal cancers are classical adenocarcinoma, and mucinous adenocarcinoma is a unique histological subtype that is known to respond poorly to chemoradiotherapy. The difference in prognosis between mucinous adenocarcinoma and classical adenocarcinoma is controversial. Here, to gain insight into the differences between classical adenocarcinoma and mucinous adenocarcinoma, we analyse 7 surgical tumour samples from 4 classical adenocarcinoma and 3 mucinous adenocarcinoma patients by single-cell RNA sequencing. Our results indicate that mucinous adenocarcinoma cancer cells have goblet cell-like properties, and express high levels of goblet cell markers (REG4, SPINK4, FCGBP and MUC2) compared to classical adenocarcinoma cancer cells. TFF3 is essential for the transcriptional regulation of these molecules, and may cooperate with RPS4X to eventually lead to the mucinous adenocarcinoma mucus phenotype. The observed molecular characteristics may be critical in the specific biological behavior of mucinous adenocarcinoma.

SPINK7 expression changes accompanied by HER2, P53 and RB1 can be relevant in predicting oral squamous cell carcinoma at a molecular level.

The oral squamous cell carcinoma (OSCC), which has a high morbidity rate, affects patients worldwide. Changes in SPINK7 in precancerous lesions could promote oncogenesis. Our aim was to evaluate SPINK7 as a potential molecular biomarker which predicts OSCC stages, compared to: HER2, TP53, RB1, NFKB and CYP4B1. This study used oral biopsies from three patient groups: dysplasia (n = 33), less invasive (n = 28) and highly invasive OSCC (n = 18). The control group consisted of clinically suspicious cases later to be confirmed as normal mucosa (n = 20). Gene levels of SPINK7, P53, RB, NFKB and CYP4B1 were quantified by qPCR. SPINK7 levels were correlated with a cohort of 330 patients from the TCGA. Also, SPINK7, HER2, TP53, and RB1, were evaluated by immunohistofluorescence. One-way Kruskal-Wallis test and Dunn's post-hoc with a p < 0.05 significance was used to analyze data. In OSCC, the SPINK7 expression had down regulated while P53, RB, NFKB and CYP4B1 had up regulated (p < 0.001). SPINK7 had also diminished in TCGA patients (p = 2.10e-6). In less invasive OSCC, SPINK7 and HER2 proteins had decreased while TP53 and RB1 had increased with respect to the other groups (p < 0.05). The changes of SPINK7 accompanied by HER2, P53 and RB1 can be used to classify the molecular stage of OSCC lesions allowing a diagnosis at molecular and histopathological levels.

Identification of Prognosis-Related Genes in Bladder Cancer Microenvironment across TCGA Database.

BACKGROUND: Bladder cancer (BCa) is a common urothelial malignancy. The Cancer Genome Atlas (TCGA) database allows for an opportunity to analyze the relationship between gene expression and clinical outcomes in bladder cancer patients. This study is aimed at identifying prognosis-related genes in the bladder cancer microenvironment. METHODS: Immune scores and stromal scores were calculated by applying the ESTIMATE algorithm. We divided bladder cancer patients into high and low groups based on their immune/stromal scores. Then, differentially expressed genes (DEGs) were identified in bladder cancer patients based on the TCGA database. We evaluated the correlation between immune/stromal scores and clinical characteristics as well as prognosis. Finally, we validated identified genes associated with bladder cancer prognosis through a cohort study in the Gene Expression Omnibus (GEO) database. RESULTS: A higher stromal score was associated with female (vs. malep = 0.037), age > 65 (vs.age ≤ 65 p = 0.015), T3/4 (vs. T1/2,p < 0.001), N status(p = 0.016), and pathological high grade (vs. low gradeP < 0.001). By analyzing DEGs, there were 1125 genes commonly upregulated, and 209 genes were commonly downregulated. Protein-protein interaction networks further showed the important protein that may be involved in the biological behavior and prognosis of BCa, such as FN1, CXCL12, CD3E, LCK, and ZAP70. A total of 14 DEGs were found to be associated with overall survival of bladder cancer. After validation by a cohort of 165 BCa cases with detailed follow-up information from GSE13507, 10 immune-associated DEGs were demonstrated to be predictive of prognosis in BCa. Among them, 5 genes have not been reported previously associated with the prognosis of BCa, including BTBD16, OLFML2B, PRRX1, SPINK4, and SPON2. CONCLUSIONS: Our study elucidated tight associations between stromal score and clinical characteristics as well as prognosis in BCa. Moreover, we obtained a group of genes closely related to the prognosis of BCa in the tumor microenvironment.

ECRG2, a novel transcriptional target of p53, modulates cancer cell sensitivity to DNA damage.

Esophageal Cancer-Related Gene 2 (ECRG2) is a recently identified tumor suppressor, its regulation and involvement in DNA damage response are unknown. Here, we show that DNA damage-induced ECRG2 upregulation coincided with p53 activation and occurred in a p53-dependent manner. We identified two p53-binding sites within ECRG2 promoter and found the promoter activity, mRNA, and protein expression to be regulated by p53. We show that DNA damage significantly enhanced p53 binding to ECRG2 promoter at the anticipated p53-binding sites. We identified a novel natural ECRG2 promoter variant harboring a small deletion that exists in the genomes of ~38.5% of world population and showed this variant to be defective in responding to p53 and DNA-damage. ECRG2 overexpression induced cancer cell death; ECRG2 gene disruption enhanced cell survival following anticancer drug treatments even when p53 was induced. We showed that lower expression of ECRG2 in multiple human malignancies correlated with reduced disease-free survival in patients. Collectively, our novel findings indicate that ECRG2 is an important target of p53 during DNA damage-induced response and plays a critical role in influencing cancer cell sensitivity to DNA damage-inducing cancer therapeutics.

Comprehensive bioinformatics analysis identifies lncRNA HCG22 as a migration inhibitor in esophageal squamous cell carcinoma.

Esophageal cancer is one of the most lethal malignancies worldwide, and esophageal squamous cell carcinoma (ESCC) is the dominant histological type. However, the long noncoding RNA (lncRNA) alterations in ESCC have not been elucidated to date. In this study, reliable databases from Gene Expression Omnibus (GEO), which analyzed lncRNA expression in ESCC tumor tissues and adjacent normal tissues were searched, and common differentially expressed lncRNAs and genes were analyzed. Next, cis- trans analysis was performed to predict the underlying relationships between altered lncRNAs and mRNAs, and the lncRNA-mRNA regulatory network was established. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of altered lncRNA-related genes were performed. The promising lncRNA HCG22 was validated by quantitative polymerase chain reaction (qPCR), and clinicopathological data were collected to identify the relationship between lncRNA HCG22 expression level and clinical features. Finally, Transwell assays were performed to explore the biological functions of lncRNA HCG22 in ESCC cells. Two hundred forty-one lncRNAs and 835 mRNAs were observed to be remarkably altered between ESCC tumor tissues and adjacent normal tissues. The lncRNA-mRNA regulatory network showed the coexpression association between lncRNA HCG22 and SPINK7 and ADAMTS12. GO and KEGG analyses showed that HCG22 and ADAMTS12 had potential biological functions in the cell migration of ESCC. The downregulation of lncRNA HCG22 in ESCC tumor tissues was validated by qPCR, and the clinicopathological data showed a noticeable correlation between lncRNA HCG22 expression level and the ESCC differentiational degree and clinical TNM stage. Kaplan-Meier analysis showed that patients with ESCC having low lncRNA HCG22 expression in ESCC tissues had considerably shorter overall survival compared with patients with ESCC having high lncRNA HCG22 expression. Following Transwell assays confirmed the migratory role of lncRNA HCG22 in ESCC cells. In conclusion, lncRNA HCG22 was downregulated in ESCC tissues and can be a migration inhibitor of ESCC cells, and SPINK7 and ADAMTS12 are promising to be the regulatory targets of lncRNA HCG22.

The Role of Serine Peptidase Inhibitor Kazal Type 13 (SPINK13) as a Clinicopathological and Prognostic Biomarker in Patients with Clear Cell Renal Cell Carcinoma.

BACKGROUND The serine peptidase inhibitor Kazal type 13 (SPINK13) gene has tumor suppressor activity, but its role in renal cell carcinoma (RCC) remains unknown. This study aimed to investigate mRNA expression of SPINK13 in clear cell renal cell carcinoma (CCRCC) in human tissue and to use bioinformatics data to investigate the role of SPINK13 expression as a clinicopathological and prognostic biomarker for patients with CCRCC. MATERIAL AND METHODS Patients with CCRCC (N=533) with available RNA sequence data from The Cancer Genome Atlas (TCGA)-CCRCC database were analyzed with patients who had a tissue diagnosis of CCRCC (N=305) at the Fudan University Shanghai Cancer Center (FUSCC). Differential transcriptional and proteome expression profiles were obtained from the ONCOMINE cancer microarray database, TCGA, and the Human Protein Atlas (HPA) database. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) measured SPINK13 mRNA expression in 305 samples of CCRCC tissue from the FUSCC. The effects of clinicopathological parameters on progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier and log-rank test. RESULTS Transcriptional and proteome expression of SPINK13 were significantly increased CCRCC tissue samples. Increased SPINK13 mRNA expression was significantly associated with reduced PFS and OS in 838 patients with CCRCC patients from the two independent cohorts, the FUSCC and the TCGA-CCRCC cohorts (p<0.01). Gene set enrichment analysis (GSEA) showed that SPINK13 expression was involved in complement, apical junction, epithelial-mesenchymal transition (EMT), glycolysis, hypoxia, and inflammation signaling pathways. CONCLUSIONS Increased expression of SPINK13 was associated with poor prognosis in patients with CCRCC.

Tazarotene-Induced Gene 1 (TIG1) Interacts with Serine Protease Inhibitor Kazal-Type 2 (SPINK2) to Inhibit Cellular Invasion of Testicular Carcinoma Cells.

Tazarotene-induced gene 1 (TIG1) encodes a protein that is a retinoid-regulated tumor suppressor. TIG1 is expressed in most normal tissues, and downregulation of TIG1 expression in multiple cancers is caused by promoter hypermethylation. Kazal-type serine protease inhibitor-2 (SPINK2) is a serine protease inhibitor, and the SPINK protein family has been shown to inhibit the expression of urokinase-type plasminogen activator (uPA). In addition, increased levels of uPA and the uPA receptor were observed in testicular cancer tissues. This study demonstrated that TIG1 interacts with SPINK2 in NT2/D1 testicular carcinoma cells. TIG1 and SPINK2 were highly expressed in normal testis tissues, while low expression levels of TIG1 and SPINK2 were found in testicular cancer tissues. TIG1 inhibited cell invasion, migration, and epithelial-mesenchymal transition (EMT) of NT2/D1 cells. SPINK2 enhanced TIG1-regulated uPA activity and EMT suppression, while silencing SPINK2 alleviated TIG1-mediated EMT regulation, cell migration, and invasion. Therefore, the results suggest that the interaction between TIG1 and SPINK2 plays an important role in the inhibition of testicular cancer cell EMT, and suppression is mediated through downregulation of the uPA/uPAR signaling pathway.

A protein scaffold, engineered SPINK2, for generation of inhibitors with high affinity and specificity against target proteases.

Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.

Novel urokinase-plasminogen activator inhibitor SPINK13 inhibits growth and metastasis of hepatocellular carcinoma in vivo.

Advanced hepatocellular carcinoma (HCC) is a highly aggressive malignancy that is a serious threat to the public health system of China. Urokinase-plasminogen activator (uPA) can promote the invasive growth and metastasis of HCC cells by activating matrix metalloproteinases (MMPs), leading to the breakage of the extra-cellular matrix. uPA is a promising target for advanced HCC treatment. In this stuy the expression of uPA was examined by quantitative polymerase chain reaction in hepatic cell lines. Protein interaction between uPA and SPINK13 was identified by immunoprecipitation. In vitro biochemical assay was used to examine the inhibitory effect of the SPINK13 on the direct cleaving of the recombinant pro-MMP9 by uPA. The antitumor effect of SPINK13 was examined by transwell assay or the nude mice tumor model.The expression of uPA was much higher in highly aggressive HCC cell lines than in lowly aggressive HCC cell lines or non-tumor hepatic cell lines. SPINK13 interacted with uPA in HCC cells and directly inhibited the cleaving of MMP9 by uPA. Treatment of the recombinant SPINK13 protein inhibited the invasion of HCC cells in several experiments, such as transwell experiments or the intrahepatic growth model. The results of the study indicated that SPINK13 could function as a promising therapeutic approach for patients with advanced HCC.

Downregulated SPINK4 is associated with poor survival in colorectal cancer.

BACKGROUND: SPINK4 is known as a gastrointestinal peptide in the gastrointestinal tract and is abundantly expressed in human goblet cells. The clinical significance of SPINK4 in colorectal cancer (CRC) is largely unknown. METHODS: We retrieved the expression data of 1168 CRC patients from 3 Gene Expression Omnibus (GEO) datasets (GSE24551, GSE39582, GSE32323) and The Cancer Genome Atlas (TCGA) to compare the expression level of SPINK4 between CRC tissues and normal colorectal tissues and to evaluate its value in predicting the survival of CRC patients. At the protein level, these results were further confirmed by data mining in the Human Protein Atlas and by immunohistochemical staining of samples from 81 CRC cases in our own center. RESULTS: SPINK4 expression was downregulated in CRC compared with that in normal tissues, and decreased SPINK4 expression at both the mRNA and protein levels was associated with poor prognosis in CRC patients from all 3 GEO datasets, the TCGA database and our cohort. Additionally, lower SPINK4 expression was significantly related to higher TNM stage. Moreover, in multivariate regression, SPINK4 was confirmed as an independent indicator of poor survival in CRC patients in all databases and in our own cohort. CONCLUSIONS: We concluded that reduced expression of SPINK4 relates to poor survival in CRC, functioning as a novel indicator.

The antiprotease SPINK7 serves as an inhibitory checkpoint for esophageal epithelial inflammatory responses.

Loss of barrier integrity has an important role in eliciting type 2 immune responses, yet the molecular events that initiate and connect this with allergic inflammation remain unclear. We reveal an endogenous, homeostatic mechanism that controls barrier function and inflammatory responses in esophageal allergic inflammation. We show that a serine protease inhibitor, SPINK7 (serine peptidase inhibitor, kazal type 7), is part of the differentiation program of human esophageal epithelium and that SPINK7 depletion occurs in a human allergic, esophageal condition termed eosinophilic esophagitis. Experimental manipulation strategies reducing SPINK7 in an esophageal epithelial progenitor cell line and primary esophageal epithelial cells were sufficient to induce barrier dysfunction and transcriptional changes characterized by loss of cellular differentiation and altered gene expression known to stimulate allergic responses (for example, FLG and SPINK5). Epithelial silencing of SPINK7 promoted production of proinflammatory cytokines including thymic stromal lymphopoietin (TSLP). Loss of SPINK7 increased the activity of urokinase plasminogen-type activator (uPA), which in turn had the capacity to promote uPA receptor-dependent eosinophil activation. Treatment of epithelial cells with the broad-spectrum antiserine protease, α1 antitrypsin, reversed the pathologic features associated with SPINK7 silencing. The relevance of this pathway in vivo was supported by finding genetic epistasis between variants in TSLP and the uPA-encoding gene, PLAU We propose that the endogenous balance between SPINK7 and its target proteases is a key checkpoint in regulating mucosal differentiation, barrier function, and inflammatory responses and that protein replacement with antiproteases may be therapeutic for select allergic diseases.

Downregulation of SPINK13 Promotes Metastasis by Regulating uPA in Ovarian Cancer Cells.

BACKGROUND/AIMS: Ovarian cancer (OC) is the fifth leading cause of cancer-related death in women, and it is difficult to diagnose at an early stage. The purpose of this study was to explore the prognostic biological markers of OC. METHODS: Univariate Cox regression analysis was used to identify genes related to OC prognosis from the Cancer Genome Atlas(TCGA) database. Immunohistochemistry was used to analyse the level of SPINK13 in OC and normal tissues. Cell proliferation, apoptosis and invasion were performed using MTT assay, flow cytometric analysis and Transwell assay, respectively. RESULTS: We identified the Kazal-type serine protease inhibitor-13 (SPINK13) gene related to OC prognosis from the Cancer Genome Atlas (TCGA) database by univariate Cox regression analysis. Overexpression of SPINK13 was associated with higher overall survival rate in OC patients. Immunohistochemistry showed that the level of SPINK13 protein was significantly lower in OC tissues than in normal tissues (P < 0.05).In vitro experiments showed that the overexpression of SPINK13 inhibited cellular proliferation and promoted apoptosis. Moreover, SPINK13 inhibited cell migration and epithelial to mesenchymal transition (EMT). SPINK13 was found to inhibit the expression of urokinase-type plasminogen activator (uPA), while recombinant uPA protein could reverse the inhibitory effect of SPINK13 on OC metastasis. CONCLUSION: These results indicate that SPINK13 functions as a tumour suppressor. The role of SPINK13 in cellular proliferation, apoptosis and migration is uPA dependent, and SPINK13 may be used as a potential biomarker for diagnosis and targeted therapy in OC.