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

Low efficacy of treatments and chemoresistance are challenges in addressing refractory hepatocellular carcinoma (HCC). SPINK1, an oncogenic protein, is frequently overexpressed in many HCC cases. However, the impact of SPINK1 on HCC treatment resistance remains poorly understood. Here, we elucidate the functions of SPINK1 on HCC therapy resistance. Analysis of SPINK1 protein level reveals a correlation between elevated SPINK1 expression and unfavorable prognosis. Furthermore, intercellular variations in SPINK1 expression levels are observed. Subsequent examination of single cell RNA-sequencing data from two HCC cohorts further suggest that SPINK1-high cells exhibit heightened activity in drug metabolic pathways compared to SPINK1-low HCC cells. High SPINK1 expression is associated with reduced sensitivities to both chemotherapy drugs and targeted therapies. Moreover, spatial transcriptomics data indicate that elevated SPINK1 expression correlates with non-responsive phenotype during treatment with targeted therapy and immune checkpoint inhibitors. This is attributed to increased levels of drug metabolic regulators, especially CES2 and CYP3A5, in SPINK1-high cells. Experimental evidence further demonstrates that SPINK1 overexpression induces the expression of CES2 and CYP3A5, consequently promoting chemoresistance to sorafenib and oxaliplatin. In summary, our study unveils the predictive role of SPINK1 on HCC treatment resistance, identifying it as a potential therapeutic target for refractory HCC.

Serum tumour associated trypsin inhibitor, as a biomarker for survival in renal cell carcinoma.

OBJECTIVE: Tumour associated trypsin inhibitor (TATI) is a peptide that is a marker for several tumours. TATI may also behave as an acute phase reactant in severe inflammatory disease. Overexpression of TATI predicts an unfavourable outcome for many cancers. This study aimed to evaluate the prognostic value of pre- and postoperative concentration of TATI in serum (S-TATI) of patients with renal cell carcinoma (RCC). MATERIALS AND METHODS: S-TATI was determined by time resolved immunofluorometric assay in preoperative and postoperative samples that were collected from 132 RCC patients, who underwent partial or complete nephrectomy in Helsinki University Hospital from May 2005 to July 2010. RESULTS: Preoperative S-TATI was significantly associated with tumour stage, lymph-node involvement, metastatic stage, Chronic Kidney Disease Stage (CKD grade), and preoperative C-reactive protein level (p < 0.05). Postoperative S-TATI was significantly associated only with CKD grade (p < 0.001). Multivariate Cox regression analysis of postoperative S-TATI, as a continuous variable, was an independent prognostic factor for overall survival (HR = 1.01, 95% CI = 1.00-1.01, p = 0.03) and cancer-specific survival (CSS) (HR = 1.01, 95% CI = 1.00-1.02, p = 0.004). CONCLUSIONS: Our data suggest that elevated postoperative S-TATI may be associated with adverse prognosis in RCC patients.

Pancreatic secretory trypsin inhibitor reduces multi-organ injury caused by gut ischemia/reperfusion in mice.

Intestinal ischemia/reperfusion (I/R) injury occurs during transplantation, mesenteric arterial occlusion, trauma and shock, causing systemic inflammation, multiple organ dysfunction and high mortality. Pancreatic secretory trypsin inhibitor (PSTI), a serine protease inhibitor expressed in gut mucosa may function as a mucosal protective/repair peptide. We examined whether PSTI affected mesenteric I/R-induced injury. Hypoxia/normoxia (H/N) caused 50% drop in cell viability of AGS, RIE1 and Caco-2 cells but PSTI (10 µg/ml) given prior- or during-hypoxic period improved survival by 50% (p<0.01). Similarly, Caco-2 monolayers exposed to H/N had 300% increase in transepithelial permeability, PSTI truncated this by 50% (p<0.01). Mice underwent mesenteric I/R by clamping jejunum, causing severe mucosal injury, increased apoptotic markers and 3-fold increases in plasma IL-6, IL1ß, TNFα, and tissue lipid peroxidation (MDA) and inflammatory infiltration (MPO) levels. Lungs showed similar significant injury and inflammatory infiltrate markers. Smaller increases in MDA and MPO were seen in kidney & liver. PSTI (20 mg/kg) reduced all injury markers by 50-80% (p<0.01). In vitro and in vivo studies showed PSTI reduced pro-apoptotic Caspase 3, 9 and Baxα levels, normalised Bcl2 and caused additional increases in HIF1α, VEGF and Hsp70 above rises caused by I/R alone (all p<0.01). PSTI also prevented reduction of tight junction molecules ZO1 and Claudin1 (all p<0.01) but did not affect increased ICAM-1 caused by I/R in gut or lung. PSTI may be a useful clinical target to prevent I/R injury.

Base variant of human pancreatic secretory trypsin inhibitor in healing of stress-induced gastric lesions in rats.

Pancreatic secretory trypsin inhibitor (PSTI) is an inhibitor of serine-proteinases including pancreatic trypsin that prevents excessive digestion of the gastrointestinal mucus, but its role in the mechanism of mucosal defense has been little studied. This study was designed to determine the effect of base variant of human PSTI (R44S-PSTI) on gastric secretion, healing of gastric lesions induced by stress and the expression of PSTI during mucosal recovery from stress lesions. Recombinant R44S-PSTI was obtained using by site-directed mutagenesis due to replacement of arginine by serine that led to longer half life of this peptide than its natural form. Stress ulcerations were induced by exposure of rats to a standard 3.5 h of water immersion and restraint stress with or without pretreatment with vehicle or R44S-PSTI (0.1 mg/kg) applied s.c. 30 min before and immediately after the end of stress. Rats were then sacrificed immediately (time 0) and at 6 h or 12 h after the termination of stress. The gastric blood flow (GBF) was measured by H2-gas clearance technique at each time period and gastric mucosal samples were excised for assessment of PSTI immunohistochemical expression and PSTI messenger RNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Southern hybridization. Stress produced numerous gastric lesions and decreased the GBF by about 30% as compared to the respective value in vehicle-treated non-stressed gastric mucosa. R44S-PSTI given s.c. in graded doses (0.01-1 mg/kg) inhibited dose-dependently gastric acid and pepsin outputs, in rats with gastric fistula and accelerated the healing of stress-induced gastric lesions significantly. The healing effects of R44S-PSTI (0.1 mg/kg s.c.) recorded at 6 h and 12 h after the end of stress were accompanied by a significant rise in the GBF. The expression of PSTI mRNA in the intact mucosa was weak, but following exposure to stress it was significantly augmented to reach the highest observed value at 6 h after the stress. We conclude that (1) base variant of human PSTI accelerates healing of stress-induced gastric lesions probably due to its antisecretory activity and enhancement of mucosal blood flow and (2) the expression of genes for PSTI plays an important role in the mechanism of mucosal recovery from gastric lesions induced by stress.

Protective effect of human pancreatic secretory trypsin inhibitor on cerulein-induced acute pancreatitis in rats.

We examined the protective effect of human pancreatic secretory trypsin inhibitor (PSTI), a specific trypsin inhibitor secreted from pancreatic acinar cells into the pancreatic duct, on cerulein-induced acute pancreatitis in conscious rats. The protective effect of human PSTI-RS, an analogue of PSTI with Arg-44 to Ser substitution which has a longer half-life in vitro, was also examined. Intraperitoneal administration of a pharmacological dose of cerulein to conscious rats induced acute pancreatitis, characterized by light microscopy as cellular disorganization of the acini and interstitial edema. Intravenous infusion of human PSTI (10, 50 or 250 micrograms/rat/h) into rats with cerulein-induced acute pancreatitis decreased their pancreatic wet weight and plasma amylase concentration. It also caused a dose-dependent decrease in vacuoles in acinar cells and interstitial edema. Human PSTI-RS, which has a longer half-life in vivo, was more effective than native PSTI at the same dose rate (10 micrograms/rat/h) in reducing pancreatitis. These results suggest that human PSTI may have a beneficial effect on acute pancreatitis.

Local administration of human pancreatic secretory trypsin inhibitor prevents the development of experimental acute pancreatitis in rats and dogs.

The objective of this investigation was to test the capacity of recombinant human pancreatic secretory trypsin inhibitor (rhPSTI) to provide prophylaxis against experimental pancreatitis. Acute hemorrhagic pancreatitis was induced by intraductal injection of sodium taurocholate in rats and by intraductal injection of bile in dogs. In one treatment group of rats the injection of taurocholate was preceded by injection of rhPSTI. In a second group of rats the rhPSTI was given intraperitoneally starting 15 min after the induction of acute pancreatitis. The survival rate in a control group of rats was 13%. In contrast, the survival rate in groups receiving rhPSTI intraductally or intraperitoneally was 80% and 63%, respectively. The survival rate in a control group of dogs was 40% at 24 h and 0% at 48 h. In contrast, all the dogs receiving a single intraductal dose of rhPSTI, either immediately before the bile injection or mixed with the bile, survived for up to 6 weeks. Detailed biochemical and immunohistologic studies in the dog indicate that, whereas rhPSTI cannot prevent the initial bile-induced injury, it does prevent the subsequent development of that injury to the point where there is massive damage to the pancreas and the surrounding tissues, and changes in blood chemistry. The development of the initial injury is, therefore, presumed to involve activation of trypsinogen. Since rhPSTI prevents the serious consequences of experimental pancreatic injury by blocking the action of trypsin, and since the pathobiochemistry of human acute pancreatitis also implies an important role for trypsin, it is possible that rhPSTI could protect humans from the pancreatitis that complicates endoscopic retrograde cholangiopancreatography and endoscopic papillotomy.