Tumor-associated Trypsin Inhibitor Research Articles

TATI, TAT-2, and CRP as Prognostic Factors in Colorectal Cancer

Objectives: Colorectal cancer is the third most common cancer worldwide, with an obvious need for more accurate prognostics. Previous studies identified C-reactive protein (CRP) as a prognostic serum biomarker for colorectal cancer, whereas the biomarkers tumor-associated trypsin inhibitor (TATI) and tumor-associated trypsin-2 (TAT-2) are less well-known prognostic factors. Therefore, in this study, we aimed to compare the prognostic role of these biomarkers. Materials and Methods: Our cohort consisted of 219 women and 274 men who underwent colorectal cancer surgery at Helsinki University Central Hospital from 1998 through 2005. Serum and plasma samples were collected before surgery, aliquoted, stored at −80°C, and then analyzed using high-sensitivity methods with commercially available time-resolved immunofluorometric assay kits. Results: In univariate analysis, CRP (HR 1.67; 95% confidence interval [CI]: 1.25–2.23; p = 0.001), TATI (HR 1.87; 95% CI: 1.13–3.08; p = 0.014), and TAT-2 (HR 1.52; 95% CI: 1.13–2.06; p = 0.006) were significant prognostic biomarkers across the entire cohort. In subgroup analyses, TATI and TAT-2 represented significant negative prognostic factors among patients older than 66, while patients with left-sided disease, a high serum TAT-2, or a high plasma CRP experienced worse prognosis. None of the biomarkers emerged as important in the disease stage subgroup analysis nor did they serve as independent factors in the multivariate analysis. Conclusions: TATI and TAT-2 as well as CRP significantly, but not independently, served as prognostic factors in our cohort of colorectal cancer patients. Further research is needed to fully understand their clinical role in colorectal cancer.

Tumor-Associated Trypsin Inhibitor (TATI) as a Biomarker of Poor Prognosis in Oropharyngeal Squamous Cell Carcinoma Irrespective of HPV Status.

Simple SummaryOropharyngeal squamous cell carcinoma (OPSCC) is a form of head and neck cancer in which human papillomavirus (HPV) infection has been shown to play a major role in disease development. The survival rates of HPV-positive patients are favorable compared to HPV-negative patients, but the reason for this phenomenon remains unclear. The management of OPSCC is complex, and development of novel treatment options is urgently required. Various possible factors affecting survival have been explored, including the tumor environment and cancer-related proteases. Our aim was to study a protease inhibitor known as tumor-associated trypsin inhibitor and its correlation with survival and clinical data in OPSCC patients.Background: We studied the role of tumor-associated trypsin inhibitor (TATI) in serum and in tumor tissues among human papillomavirus (HPV)-positive and HPV-negative OPSCC patients. Materials and methods: The study cohort included 90 OPSCC patients treated at the Helsinki University Hospital (HUS), Helsinki, Finland, in 2012–2016. TATI serum concentrations (S-TATIs) were determined by an immunofluorometric assay. Immunostaining was used to assess tissue expression. HPV status was determined with a combination of p16 immunohistochemistry and HPV DNA PCR genotyping. The survival endpoints were overall survival (OS) and disease-specific survival (DSS). Results: A significant correlation was found between S-TATI positivity and poor OS (p < 0.001) and DSS (p = 0.04) in all patients. In HPV-negative cases, S-TATI positivity was linked to poor OS (p = 0.01) and DSS (p = 0.05). In HPV-positive disease, S-TATI positivity correlated with poor DSS (p = 0.01). S-TATI positivity was strongly associated with HPV negativity. TATI serum was negatively linked to a lower cancer stage. TATI expression in peritumoral lymphocytes was associated with favorable OS (p < 0.025) and HPV positivity. TATI expression in tumor and in peritumoral lymphocytes correlated with lower cancer stages. Conclusion: Our results suggest that S-TATI positivity may be a biomarker of poor prognosis in both HPV-positive and HPV-negative OPSCC.

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.

Diagnostic and Prognostic Potential of Biomarkers CYFRA 21.1, ERCC1, p53, FGFR3 and TATI in Bladder Cancers.

The high occurrence of bladder cancer and its tendency to recur in combination with a lifelong surveillance make the treatment of superficial bladder cancer one of the most expensive and time-consuming. Moreover, carcinoma in situ often leads to muscle invasion with an unfavorable prognosis. Currently, invasive methods including cystoscopy and cytology remain a gold standard. The aim of this study was to explore urine-based biomarkers to find the one with the best specificity and sensitivity, which would allow optimizing the treatment plan. In this review, we sum up the current knowledge about Cytokeratin fragments (CYFRA 21.1), Excision Repair Cross-Complementation 1 (ERCC1), Tumour Protein p53 (Tp53), Fibroblast Growth Factor Receptor 3 (FGFR3), Tumor-Associated Trypsin Inhibitor (TATI) and their potential applications in clinical practice.

Tumor-associated trypsin inhibitor (TATI) and tumor-associated trypsin-2 (TAT-2) predict outcomes in gastric cancer

Introduction: Tumor-associated trypsin inhibitor (TATI) limits serine proteases, promotes carcinogenesis in several cancers and functions as an acute-phase reactant. Tumor-associated trypsin-2 (TAT-2), a proteolytic target enzyme for TATI, can enhance invasion by promoting extracellular matrix degradation. Here, we aimed to study serum TATI and TAT-2 levels, including the TAT-2/TATI ratio, as prognostic and diagnostic biomarkers in gastric cancer. We compared the results with the plasma level of C-reactive protein (CRP).Material and Methods: We selected 240 individuals operated on for gastric adenocarcinoma at the Helsinki University Hospital, Finland, between 2000 and 2009. We determined the preoperative serum TAT-2, TATI and plasma CRP levels using time-resolved immunofluorometric assays using monoclonal antibodies.Results: The medium serum TAT-2 level was higher among gastric cancer patients [8.68 ng/ml; interquartile range (IQR) 5.93–13.2] than among benign controls (median 5.41 ng/ml; IQR 4.12–11.8; p = .005). Five-year survival among patients with a high serum TAT-2 was 22.9% [95% confidence interval (CI) 11.7–34.1], compared to 52.2% (95% CI 44.6–59.8; p < .001) among those with a low level. The five-year survival among patients with a high serum TATI was 30.6% (95% CI 20.4–40.8), compared to 52.9% (95% CI 44.7–61.1; p < .001) among those with a low level. The serum TATI level remained significant in the multivariable survival analysis (hazard ratio 2.01; 95% CI 1.32–3.07). An elevated plasma CRP level associated with a high serum TATI level (p = .037).Conclusions: This study shows for the first time that a high serum TAT-2 may function as a prognostic biomarker in gastric cancer and that TAT-2 levels may be elevated compared to controls. Additionally, we show that the prognosis is worse among gastric cancer patients with a high serum TATI. These biomarkers serve as prognostic factors particularly among patients with a metastatic or a locally advanced disease.

Prognostic value of the combined expression of tumor-associated trypsin inhibitor (TATI) and p53 in patients with bladder cancer undergoing radical cystectomy.

Urothelial carcinoma of the bladder is a heterogeneous disease for which reliable prognostic molecular biomarkers have not been established. To investigate the prognostic value of tumor-associated trypsin inhibitor (TATI) expression combined with p53 expression in bladder cancer patients who have undergone radical cystectomy. Tissue microarrays from 110 patients were analyzed immunohistochemically for TATI and p53 protein expression. Complete clinical-pathological information and follow-up data were collected. Univariable Kaplan-Meier analysis and log-rank test were performed to assess the association between TATI and p53 expression patterns with clinical outcomes. Cox's proportional hazard analysis was performed to identify potential independent risk factors for predicting disease progression and evaluate the prognostic value of combining the expression of TATI and p53 on progression-free survival (PFS) and overall survival (OS). TATI expression was positively correlated with favorable differentiation of bladder cancer, and lower tumor stage. p53 expression was positively related to tumor stage, tumor grade, and lymph-node invasion. Univariate Kaplan-Meier analysis revealed significant differences between TATI-positive vs. TATI-negative and p53-positive vs. p53-negative patients, regarding PFS. Multivariate analysis showed that both TATI and p53 expression were independent factors for predicting disease progression. TATI expression patterns could enhance the prognostic value of p53 overexpression on progression.

Differential expression of Annexin 2, SPINK1, and Hsp60 predict progression of prostate cancer through bifurcated WHO Gleason score categories in African American men.

Although studies have observed several markers correlate with progression of prostate cancer (PCa), no specific markers have been identified that accurately predict the progression of this disease, even in African American (AA) men who are generally at higher risk than other ethnic groups. The primary goal of this study was to explore whether three markers could predict the progression of PCa. We investigated protein expression of Annexin 2 (ANX2), serine peptidase inhibitor, kazal type 1(SPINK1)/tumor-associated trypsin inhibitor (TATI), and heat shock protein 60 (Hsp60) in 79 archival human prostate trans-rectal ultrasound (TRUS) biopsy tissues according to a modified World Health Organization (WHO) classification: normal (WHO1a), Gleason Score (GS6 (WHO1b), GS7 subgroups (WHO2 = 3 + 4, WHO3 = 4 + 3), GS8 (WHO4), and GS9-10 (WHO5). AA men aged 41-90 diagnosed from 1990 to 2013 at Howard University were included. Automated staining assessed expression of each biomarker. Spearman correlation assessed the direction and relationship between biomarkers, WHO and modified WHO GS, age, and 5-year survival. A two-tailed t-test and ANOVA evaluated biomarkers expression in relationship to WHO normal and other GS levels, and between WHO GS levels. A logistic and linear regression analysis examined the relationship between biomarker score and WHO GS categories. Kaplan-Meier curves graphed survival. ANX2 expression decreased monotonically with the progression of PCa while expression of SPINK1/TATI and Hsp60 increased but had a more WHO GS-specific effect; SPINK1/TATI differed between normal and GS 2-6 and HSP60 differed between GS 7 and GS 2-6. WHO GS was found to be significantly and negatively associated with ANX2, and positively with SPINK1/TATI and Hsp60 expression. High SPINK1/TATI expression together with the low ANX2 expression at higher GS exhibited a bi-directional relationship that is associated with PCa progression and survival. Importantly, the data reveal that ANX2, and SPINK1/TAT1 highly associate with WHO GS and with the transition from one stage of PrCa to the next in AA men. Future research is needed in biracial and larger population studies to confirm this dynamic relationship between ANX2 and SPINK1 as independent predictors of PCa progression in all men.

Postoperative CEA is a better prognostic marker than CA19-9, hCGβ or TATI after resection of colorectal liver metastases.

Liver metastases of colorectal cancer can be operated with a curative intent in selected cases. However, more than half of the patients have a recurrence. The aim of this study was to evaluate the prognostic and predictive value of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), human chorionic gonadotropin β (hCGβ) and tumour-associated trypsin-inhibitor (TATI) in colorectal cancer patients before and 3 months after resection of liver metastases. Marker concentrations were determined in blood samples from 168 colorectal cancer patients, who underwent liver resection between the years 1998 and 2007 at Helsinki University Hospital, Finland. The samples were taken before and 3 months after curative resection. Increased concentrations of CEA (>5 µg/L) and hCGβ (>1 pmol/L) 3 months after liver resection correlated with recurrence and impaired overall survival and increased CA19-9 (>26 kU/L) with impaired overall survival, but postoperative TATI was not prognostic. Preoperatively elevated CEA and CA19-9 correlated with impaired overall survival, but not with recurrence. Neither preoperative hCGβ nor TATI was prognostic. In conclusion, CEA is a useful prognostic marker, when measured 3 months after resection of colorectal liver metastases. CA19-9 also has prognostic significance and may have additional value.

A preliminary study of screening bile tumor markers for extrahepatic cholangiocarcinoma with proteomics technology

Objective To analyze differentially expressed proteins in bile of extrahepatic cholangiocarcinoma (EHCC)by proteomics technology, and to detect the potential tumor markers for diagnosis of bile duct malignancy. Methods Bile samples from 16 patients with EHCC and 16 patients with sphincter of oddi dysfunction (control) were collected for proteomic analysis by isobaric tags for relative and absolute quantitation (iTRAQ). The differentially expressed proteins were identified by mass spectrometry and retrieved by protein database. Results Protein maps of both EHCC group and control group were established successfully by iTRAQ. Analysis software identified an average of 275 proteins in EHCC group and 218 proteins in control group. Statistical filter screened 12 proteins of more than double difference, 4 of which were of diagnostic value, identified by mass spectrometry, which were MUC3a, Annexin A3, REG3A and Tumor-associated trypsin inhibitor (TATI). Functional analysis revealed that these proteins were associated with cancer cellular oncogenesis, proliferation, differentiation and metastasis. Conclusion Proteomic analysis by iTRAQ can identify the protein variance in bile of patients with EHCC, and identify four new potential biomarkers correlated with biological behavior of cholangiocarcinoma. Key words: Cholangiocarcinoma; Bile ducts, extrahepatic; Proteomics; Bile; Tumor markers

Abstract A42: Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer

Abstract Inflammation is known to promote colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) is primarily secreted by the cells of the tumor microenvironment (TME), mainly by inflammatory cells but also by activated fibroblasts. IL-6 stimulates growth and survival signaling in CRC. Inflammatory signals regulate also the production and activity of proteases and their inhibitors. Serine protease inhibitor Kazal type1 (SPINK1, aka tumor-associated trypsin inhibitor, TATI) is expressed in several tissues and over-expression of SPINK1 predicts an unfavorable outcome in many cancers, including colon cancer. The SPINK1 gene contains an IL-6 responsive element and in addition to being a protease inhibitor, it also acts as an acute phase reactant and a growth factor. Expression of serine proteases trypsin-1 and -2, the main targets of SPINK1, also correlate with malignancy and metastatic potential. The aim of this study was to assess the paracrine signaling between fibroblast-derived IL-6 and cancer cell-derived SPINK1, trypsin-1 and -2. The following expression analyses were used: quantitative PCR, immunohistochemistry, immunofluorometric assays and Western blotting. The results show that CRC cells Colo205 and HT-29 express SPINK1 and secrete it into the culture medium. IL-6 dose-dependently increased the mRNA expression and protein levels of SPINK1. Conditioned media from fibroblasts had the same effect, and conversely CRC media increased IL-6 secretion in the fibroblasts, indicating paracrine signaling between these cell types. In CRC tissues cancer cells were positive for SPINK1, while IL-6 was found in fibroblasts surrounding the cancer cells, confirming the in vitro results. In Colo205 cells the baseline levels of trypsin-1 and -2 and the respective genes PRSS1 and PRSS2 were much higher compared to HT-29 cells. In Colo205 cells IL-6 led to concomitant increase in the secretion of trypsin-1 and -2, whereas in HT-29 cells these remained constantly low. Mechanistically, addition of IL-6 led to activation of the canonical Stat3 pathway, as indicated by Stat3 phosphorylation. Stat3 inhibitor reduced both SPINK1 and trypsin-1 and -2 levels, demonstrating that Stat3 is the transcription factor driving their expression. Taken together, our results show a connection between TME-derived inflammatory response and increased SPINK1 and trypsin-1 and -2 levels. Elevated serum SPINK1 has been shown to be an independent prognostic factor in colon cancer. As SPINK1 acts as a growth factor in some cancers, it has also been suggested as a therapeutic target. Therefore assessment of SPINK1 expression and function has several potentially important clinical applications in colorectal and other cancers. The study also strengthens the role of tumor microenvironment in tumor progression and emphasizes the importance of studying tumor-stroma crosstalk of proteolytic processing. Citation Format: Kati Räsänen, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Outi Itkonen, Ulf-Håkan Stenman, Hannu Koistinen. Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A42.

Monitoring the treatment outcome in endometrial cancer patients by CEA and TATI.

An attempt was made to compare the usefulness of determining markers carcinoembryonic antigen (CEA) and tumor-associated trypsin inhibitor (TATI) in endometrial cancer patients in whom recurrence or distant metastasis was diagnosed in observation after treatment. The study included 316 patients aged 32–81, average age of 61 years, SD = 8.72, with diagnosed endometrial cancer, treated between 1994 and 1995 at the Oncology Center in Warsaw and then under observation from 4 months to 17 years after completion of treatment. The levels of the markers TATI and CEA were assessed from the first five serum samples taken during postoperative radiotherapy and in the initial period of observation after completed treatment. Receiver operating characteristic (ROC) curves were generated, determining the sensitivity and specificity of both CEA and TATI in patients who experienced treatment failure, i.e., recurrence and distant metastasis. Assessing the sensitivity of the marker CEA, it was found that if in the third sample, i.e., during radiation therapy, the marker level increased by more than 20 % compared with the first sample, then recurrence of cancer occurred during the observation period in 75.9 % of patients and metastatic occurred in 69.7 % of patients. In the evaluation of the marker TATI, it was found that if the level of TATI between the first and the third sample increases by 10.6 % from the initial level, then in 84.4 % (sensitivity) of cases, this means the occurrence of cancer recurrence and in 75.7 % (sensitivity) of cases, the occurrence of metastasis. The specificity of both markers is low and not useful diagnostically.

Tumor-associated trypsin inhibitor in patients with endometrial cancer.

The aim of the study was to look for prognostic factors of metastasis or recurrence in patients with endometrial cancer. Tumor-associated trypsin inhibitor (TATI) concentrations were measured in serum of 317 patients with endometrial cancer. The assay was done 7 times in each patient, from the moment of diagnosis until the start of follow-up after the completion of treatment. Observation of patients after treatment lasted from 0 to 16 years. The TATI levels in patients with adverse prognostic factors accumulated in the first 3 assays and then decreased to zero. Mean TATI concentrations were significantly higher in patients with clinically advanced disease (stage IIIB) than patients at stage I (Kruskal-Wallis p = 0.0446). An increase in the concentration by more than 10.6% in the first 3 assays was significantly correlated with disease relapse (Mann-Whitney Z = -6.06653, p = 0.00000) and local or distant recurrence (Mann-Whitney Z = -4.97475, p = 0.000001). A significant increase in the TATI level in the first 3 tests also occurred in patients who died during the study period (Kruskal Wallis p&lt;0.001). In our series of patients with endometrial cancer, TATI proved to be a sensitive indicator of disease recurrence and distant metastasis, with a sensitivity of 84.4% and 75.7%, respectively. TATI seems to behave as a prognostic factor in certain subgroups of patients with endometrial cancer.

Abstract 439: Interleukin-6 induces SPINK1 in colorectal cancer

Abstract Inflammation is known to promote colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates growth and survival signaling in CRC cells. Inflammatory signals regulate also the production and activity of proteases and their inhibitors. Serine protease inhibitor Kazal type1 (SPINK1, aka tumor-associated trypsin inhibitor, TATI) is expressed in several tissues and over-expression of SPINK1 predicts an unfavorable outcome in many cancers, including colon cancer. The SPINK1 gene contains an IL-6 responsive element and in addition to being a protease inhibitor, it also acts as an acute phase reactant and a growth factor. The aim of this study was to assess the connection between IL-6 and SPINK1 and the effect of this signaling to growth and invasion of CRC cells. The following expression analyses and functional assays were used: quantitative PCR, immunohistochemistry, immunofluorometric assays, Western blotting, cell proliferation and migration assays. The results of this study show that CRC cell lines Colo205 and HT-29 express SPINK1 and secrete it into the culture medium. In Colo205 and HT-29 cells IL-6 dose-dependently increased the mRNA expression and protein levels of SPINK1. The baseline secretion was lower in the Colo205 cells, and significantly more IL-6 was required for the dose-dependent induction of SPINK1 expression in these cells as compared to HT-29 cells. Interestingly, in Colo205 the baseline levels of secreted tumor-associated trypsinogen-1 and -2 (TAT-1 and -2), the key targets of SPINK1 protease inhibitory action, were much higher than in HT-29 cells. In these cells IL-6 led to concomitant increase in the secretion of TAT-1 and -2, whereas in HT-29 cells the TAT-1 and -2 levels remained constantly low. IL-6 at concentrations giving the maximum SPINK1 induction did not increase the proliferation of Colo205 or HT-29 cells; however the motility of these cells was increased in response to this proinflammatory cytokine. Mechanistically, addition of IL-6 led to activation of the canonical signal transducer and activator of transcription 3 (Stat3) pathway, as indicated by Stat3 phosphorylation in both Colo205 and HT-29 cells. Taken together, our results indicate a connection between inflammatory response and increased SPINK1 levels. Elevated serum SPINK1 has been shown to be an independent prognostic factor in colon cancer. As SPINK1 acts as a growth factor in some cancers, it has also been suggested as a therapeutic target. Therefore assessment of SPINK1 expression and function has several potentially important clinical applications in colorectal and other cancers. Citation Format: Kati A. Räsänen, Laura Hautala, Ulf-Håkan Stenman, Hannu Koistinen. Interleukin-6 induces SPINK1 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 439. doi:10.1158/1538-7445.AM2015-439

Overexpression of Tumor-Associated Trypsin Inhibitor (SPINK1/TATI) in Hepatitis C-Associated Hepatocellular Carcinoma: Potential Implications for Viral Hepatocarcinogenesis

Background: The molecular pathomechanisms leading to hepatitis C virus (HCV)-induced hepatocarcinogenesis remain unclear. This study investigated the molecular pathways and key genes underlying HCV-positive hepatocellular carcinoma (HCC) using gene expression profiling. Methods: Oligonucleotide arrays (Affymetrix HU133A) were used to determine and compare the tissue-specific gene expression profiles in 39 cases of HCV-positive or -negative HCC and non-malignant liver tissue. The expression values of the most overexpressed genes were validated by real-time polymerase chain reaction (RT-PCR). Results: 837 genes or expressed sequence tags (ESTs) were significantly differently expressed in HCV-positive HCC versus healthy tissue: 414 were upregulated and 423 were downregulated (p < 0.05; > 2-fold change in ≥ 70% of the samples). A specific gene expression profile for HCV-positive HCC was obtained using 2-dimensional cluster analysis and was confirmed using supervised neuronal network modeling. The most consistently overexpressed gene coded for serine protease inhibitor Kazal-type 1 (SPINK1)/tumor-associated trypsin inhibitor (TATI) (median fold change, 19.7; significantly overexpressed in 90% of the samples). SPINK1/TATI was coregulated with matrix metalloproteinases (MMPs) and their natural inhibitors (tissue inhibitors of metalloproteinases (TIMPs)). Conclusions: Gene expression profiling identified specific dysregulated molecular pathways and SPINK1/TATI as the most overexpressed gene in HCV-positive HCC. These data highlight the importance of SPINK1/TATI as a tumor marker for HCV-induced HCC and may lead to a better understanding of HCV-induced hepatocarcinogenesis.

Abstract 5304: Inhibition of kallikrein-related peptidase-4 by SPINK1

Abstract Serine protease inhibitor Kazal type-1 (SPINK1), also known as TATI (tumor-associated trypsin inhibitor), is a 56 amino acid trypsin inhibitor, whose levels are elevated in several cancers, including those of ovaries, prostate, bladder and colon. Some mutations in SPINK1, such as N34S, are associated with pancreatitis. Furthermore, SPINK1 was recently found to be associated with ETS rearrangement-negative prostate cancers and to stimulate the invasion of prostate cancer cells, perhaps by activating the EGF-receptor. Recently, SPINK3, the mouse ortholog of SPINK1, was identified as a potent inhibitor of human kallikrein-related peptidases (KLKs) -2, -3 (PSA) and -5. We studied the effect of human SPINK1 on KLK2, -3 and -4. Synthetic cDNAs encoding wild-type and mutant forms of 56 amino acid SPINK1 without signal peptide were expressed in E. coli. The SUMOstar E. coli Expression System was used for expression and purification of recombinant SPINK1. Final purification was done using an antibody affinity column and HPLC. Enzyme activities were measured using chromogenic substrates and recombinant human KLK2 and -4, and KLK3 purified from seminal fluid. SPINK1 was found to efficiently inhibit KLK4, while KLK2-inhibition was less pronounced. KLK3 was not inhibited at all. SPINK1 mutant (K41Y), which does not inhibit trypsins, did not inhibit KLK4 either, while pancreatitis associated mutant (N34S) showed strong inhibition. It is possible that SPINK1 has a dual role in cancer, i.e., it induces tumor growth by stimulating the EGF-receptor, while by inhibiting the activity of trypsin and KLKs it suppress metastasis and activation of protease-activated receptors (PARs), which promote tumor growth. Citation Format: Hannu Koistinen, Mykola Domanskyy, Ulf-Håkan Stenman. Inhibition of kallikrein-related peptidase-4 by SPINK1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5304. doi:10.1158/1538-7445.AM2014-5304

Roles of Serine Protease Inhibitor Kazal type 1 (SPINK1) in Prostate Cancer

Altered genes that play a driving role in cancer development can often serve as specific diagnostic markers, criteria of molecular classification and therefore potential therapeutic targets. Serine protease inhibitor Kazal type 1 (SPINK1), also known as pancreatic secretory trypsin inhibitor or tumor-associated trypsin inhibitor, encodes a 56 amino acid secreted peptide, and its normal function is thought to be the inhibition of serine proteases such as trypsin. Recent studies have indicated marked overexpression of SPINK1 defines an aggressive molecular subtype of ETS (erythroblastosis virus E26 transformation-specific) fusion-negative prostate cancer ((PCa) patients. SPINK1 may act as an autocrine growth factor and promotes PCa growth and invasion. Most recently, we suggested that SPINK1 induces epithelial-mesenchymal transition (EMT) through EGFR signaling pathway in PCa. The association between SPINK1 overexpression and poor prognosis in PCa has been reported. Notably, SPINK1 might be a novel extracellular therapeutic target in a subset of high-grade PCa patients. In this review, we will summarize the current understanding of SPINK1 involving its role in PCa biology, association with prognosis as well as perspective in therapy from the pathologist's point of view.

A novel discriminant score based on tumor-associated trypsin inhibitor for accurate diagnosis of metastasis in patients with breast cancer

Invasion and metastasis of solid tumors require proteolytic enzymes for degradation of the basal membrane and extracellular matrix. Currently, there are no reliable methodologies to predict the risk for metastatic disease. In this context, our aim has been focused on the development of a noninvasive score based on tumor-associated trypsin inhibitor (TATI) for the assessment of metastasis in patients with breast cancer. TATI, trypsin, and soluble epidermal growth factor receptor (sEGFR) were assayed by enzyme-linked immunosorbent assay. CA 15.3 serum level was assayed by microparticle enzyme immunoassay in 265 patients with breast cancer. Statistical analyses were performed by logistic regression and receiver operating characteristic analysis curves. Using multivariate discriminant analysis, a score is selected based on absolute values of the four biochemical markers: TATI-metastatic breast cancer score (TATI-MBCS) = [0.03 × CA 15.3 (U/L) + 0.039 × TATI (ng/ml) + 0.04 × trypsin (ng/ml) + 0.023 × sEGFR (ng/ml) - 6.49 (numerical constant)]. This function correctly classified 84% of metastatic breast cancer at cutoff value = 0.62 (i.e., greater than 0.62 indicates patients with metastatic breast cancer and less than 0.62 indicates patients with nonmetastatic breast cancer). In conclusion, TATI-MBCS is a novel, noninvasive, and simple score which can be applied to discriminate patients with metastatic breast cancer.

Prognostic significance of tumor-associated trypsin inhibitor (TATI) and human chorionic gonadotropin-β (hCGβ) in patients with hepatocellular carcinoma

Aim. Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer death worldwide. The aim of this study was to evaluate the prognostic value of serum tumor-associated trypsin inhibitor (TATI) and the free β subunit of human chorionic gonadotropin (hCGβ) in patients with HCC. Methods. The serum concentrations of TATI and hCGβ were determined by time-resolved immunofluorometric assays (IFMA) in pretreatment serum samples from 144 patients with HCC. Clinical data were retrieved from patient records and survival data obtained from Statistics Finland. Results. The overall cumulative disease-specific survival was 69% at 1 year, 50% at 2 years and 33% at 5 years. Disease-specific median survival time was 26 months. The overall survival in patients with low serum concentrations of TATI or hCGβ was statistically significantly better than in patients with elevated concentrations (p = 0.003 and 0.003, respectively). In multivariate analysis, both serum TATI and serum hCGβ were independent prognostic markers. Conclusion. The results imply that elevated serum concentrations of TATI and hCGβ are predictors of adverse prognosis in patients with HCC and appear to be useful adjuncts in predicting prognosis in patients with HCC.

A high-density tissue microarray from patients with clinically localized prostate cancer reveals ERG and TATI exclusivity in tumor cells

Background:Prostate cancer (PCa) is characterized by high tumor heterogeneity. In 2005, the fusion between the androgen-regulated gene TMPRSS2 and members of the ETS family was discovered in prostate cancer. In particular, fusion of TMPRSS2 with ERG was found in approximately 50% of prostate cancers and considered as an early event in the onset of the disease. The prognostic value of this fusion is still contradictory. Bioinformatics showed that overexpression of SPINK1 gene in a subset of fusion-gene-negative prostate cancers was associated with a poor prognosis. In theory, overexpression of the tumor-associated trypsin inhibitor (TATI) protein encoded by SPINK1 in fusion-gene-negative tumor cells opens the way to selected treatments for genotypically different cases. However, their expression has never been assessed at the cellular level in the same tissue samples.Methods:As ERG expression has been shown to be a surrogate of fusion gene occurrence in prostate cancer, we have used double immunohistochemical staining to assess expression of ERG and TATI on a large tissue microarray comprising 4177 cases of localized prostate cancer.Results:We did not detect any co-expression of ERG and TATI in the same cancer cells, which confirms previous suggestions from in silico studies. ERG was associated with Gleason score (GS), surgical margins and pathological stage, but had no prognostic value in this cohort. TATI was weakly associated with pathological stage but had no significant association with outcome.Conclusions:We here provide a morphological basis for ERG and TATI exclusivity in prostate cancer cells. Future therapies should be based on a combination of different targets in order to eradicate tumor cells with gene fusions and cells expressing other tumor-associated antigens. Further studies are needed to understand why ERG and TATI are not co-expressed in the same prostatic tumor cells.

Su1251 Overexpression of EGFR in the Exocrine Pancreas Induces a Chronic Pancreatitis

Background and Aim: Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) frequently overexpress epidermal growth factor receptor (EGFR) and EGF-family ligands. Although chronic pancreatitis is a risk factor for PDA, the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis in development of PDA is the lack of appropriate animal models for chronic pancreatitis with similar clinical courses as in human patients. Serine protease inhibitor Kazal type 1 (SPINK1: mouse homolog Spink3), which is also known as tumor associated trypsin inhibitor (TATI), is mainly produced in the pancreatic acinar cells. Recently, we showed that SPINK1 binds to EGFR to activate its downstream signaling. The present study aims to generate chronic pancreatitis model mice and assess the molecular events from chronic pancreatitis to PDA. Methods: 1) C57BL/6 female mice were repeatedly induced acute pancreatitis by using choline-deficient with ethionine (CDE) diet for 54 weeks. 2) To assess the molecular events caused by long term CDE diet treatment using Western blotting. 3) To further analyze the clinical importance of EGFR and SPINK1 in the chronic pancreatitis and PDA, we examined the expression of SPINK1 and EGFR. Immunohistochemical analyses were performed using human chronic pancreatitis and PDA samples. Results: 1) Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 wk of CDE-diet treatment. In spite of the severe pancreatic degeneration, neoplastic changes were not observed. 2) Expression of EGFR and its ligands (Spink3 and TGF α) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. In addition, cyclooxygenase-2 which induced by stimuli involved in inflammatory responses was also overexpressed. 3) In the human chronic pancreatitis samples, TCs were positive EGFR immunostaining. Furthermore, PDA cases were positive for both SPINK1 and EGFR immunoreactivity. Conclusion: We have successfully developed a chronic pancreatitis model with CDE diet. We also showed expression of EGFR and its potent ligands, phosphorylation of EGFR, and activation of K-Ras as the step of chronic inflammation. It was suggested that EGFR signaling cascades were important for the development from chronic pancreatitis to PDA. Howevere, chronic pancreatitis itself is not sufficient for the development of PDA. Further combinations of CDE-diet treatment with the administration of other promotion insults or genetic alterations may result in PDA development.