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Ulf-håkan Stenman - One of the best experts on this subject based on the ideXlab platform.
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An immunocapture-LC-MS-based assay for serum SPINK1 allows simultaneous quantification and detection of SPINK1 variants.
Analytical and bioanalytical chemistry, 2018Co-Authors: Suvi Ravela, Mykola Domanskyy, Hannu Koistinen, Ulf-håkan Stenman, Jakob Stenman, Leena Valmu, Leena Kylänpää, Outi Lindström, Esa Hämäläinen, Outi ItkonenAbstract:Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5–1000 μg/L. The limit of quantitation (LOQ) was 0.5 μg/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98–1.01, n = 11), 0.55 (95% CI 0.43–0.61, n = 14), and 0.05 (range 0.04–0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.
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MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma.
Molecular oncology, 2017Co-Authors: Kati Räsänen, Hannu Koistinen, Ulf-håkan Stenman, Caj Haglund, Kien Xuan Dang, Harri Mustonen, Susanna Lintula, Jakob StenmanAbstract:The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.
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Abstract A42: Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer
Cell-Cell Signaling in the Tumor Microenvironment, 2016Co-Authors: Kati Räsänen, Outi Itkonen, Ulf-håkan Stenman, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Hannu KoistinenAbstract: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 Rasanen, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Outi Itkonen, Ulf-Hakan 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.
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Emerging Roles of SPINK1 in Cancer
Clinical chemistry, 2016Co-Authors: Kati Räsänen, Hannu Koistinen, Outi Itkonen, Ulf-håkan StenmanAbstract:BACKGROUND: Tumor-associated trypsin inhibitor (TATI) was originally isolated from the urine of a patient with ovarian cancer. It was later shown to be produced by many other tumors and several normal tissues. It had earlier been isolated from the pancreas and was hence called pancreatic secretory trypsin inhibitor (PSTI). It belongs to a family of protease inhibitors presently called serine peptidase inhibitor Kazal type (SPINK). In the SPINK family TATI/PSTI is SPINK1, which is the name used in this review. CONTENT: In addition to being a protease inhibitor, SPINK1 also acts as an acute-phase reactant and a growth factor. Furthermore, it has been shown to modulate apoptosis. Overexpression of SPINK1 predicts an unfavorable outcome in several cancers and determination of SPINK1 in serum can be used to identify patients at increased risk of aggressive disease. Thus serum SPINK1 can be used as a prognostic tumor marker. Because SPINK1 acts as a growth factor and an inhibitor of apoptosis in some cancers, it has also been suggested that it can be a therapeutic target in cancer. However, because SPINK1 is the major physiological inhibitor of trypsin, inhibition of SPINK1 may increase the risk of pancreatitis. SUMMARY: Taking into account the many functions of SPINK1, assessing the role of SPINK1 in cancer has several potentially important clinical applications ranging from a biomarker to a potential new target for cancer therapy.
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Interleukin-6 increases expression of serine protease inhibitor Kazal type 1 through STAT3 in colorectal adenocarcinoma.
Molecular carcinogenesis, 2015Co-Authors: Kati Räsänen, Outi Itkonen, Ulf-håkan Stenman, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Teijo Kuopio, Hannu KoistinenAbstract:Inflammation promotes colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates survival signaling in CRC; inflammatory signals also regulate production and activity of proteases and their inhibitors. Over-expression of serine protease inhibitor Kazal type 1 (SPINK1) predicts an unfavorable outcome in colon cancer. The SPINK1 gene contains an IL-6 responsive element, suggesting it could act as an acute phase reactant. We assessed the connection between IL-6 and SPINK1, and the function and mechanism of this signaling. Our results show that Colo205 and HT-29 cells express and secrete SPINK1, and both fibroblast-derived and recombinant IL-6 further increased the SPINK1 levels. Concurrently CRC cells augmented the IL-6 production in fibroblasts. In CRC tissues cancer cells were positive for SPINK1, whereas IL-6 was found in stromal cells. In Colo205 cells IL-6 also stimulated the secretion of trypsin-1 and -2, the key targets of SPINK1 protease inhibition, whereas in HT-29 cells trypsin-1 and -2 levels remained constantly low. Functionally, both IL-6 and SPINK1 increased the motility of the CRC cells. Mechanistically, IL-6 activated the canonical STAT3 pathway and inhibition of STAT3 phosphorylation decreased the levels of SPINK1, trypsin-1 and -2. Taken together, our results indicate a novel link between inflammatory signals originating from the tumor microenvironment and increased SPINK1 levels. This finding has potential therapeutic implications for targeted therapy, as it confirms that SPINK1 acts as an acute phase reactant and that it participates in the paracrine crosstalk with the tumor microenvironment of colon cancer. © 2015 Wiley Periodicals, Inc.
Jian-min Chen - One of the best experts on this subject based on the ideXlab platform.
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Toward a clinical diagnostic pipeline for SPINK1 intronic variants
Human Genomics, 2019Co-Authors: Xin-ying Tang, Wen-bin Zou, Zhuan Liao, Claude Férec, Emmanuelle Masson, David Cooper, Jin-huan Lin, Arnaud Boulling, Shun-jiang Deng, Jian-min ChenAbstract:Background: The clinical significance of SPINK1 intronic variants in chronic pancreatitis has been previously assessed by various approaches including a cell culture-based full-length gene assay. A close correlation between the results of this assay and in silico splicing prediction was apparent. However, until now, a clinical diagnostic pipeline specifically designed to classify SPINK1 intronic variants accurately and efficiently has been lacking. Herein, we present just such a pipeline and explore its efficacy and potential utility in potentiating the classification of newly described SPINK1 intronic variants. Results: We confirm a close correlation between in silico splicing prediction and results from the cell culture-based full-length gene assay in the context of three recently reported pathogenic SPINK1 intronic variants. We then integrated in silico splicing prediction and the full-length gene assay into a stepwise approach and tested its utility in the classification of two novel datasets of SPINK1 intronic variants. The first dataset comprised 16 deep intronic variants identified in 52 genetically unexplained Chinese chronic pancreatitis patients by sequencing the entire intronic sequence of the SPINK1 gene. The second dataset comprised five novel rare proximal intronic variants identified through the routine analysis of the SPINK1 gene in French pancreatitis patients. Employing a minor allele frequency of > 5% as a population frequency filter, 6 of the 16 deep intronic variants were immediately classified as benign. In silico prediction of the remaining ten deep intronic variants and the five rare proximal intronic variants with respect to their likely impact on splice site selection suggested that only one proximal intronic variant, c.194 + 5G > A, was likely to be of functional significance. Employing the cell culture-based full-length gene assay, we functionally analyzed c.194 + 5G > A, together with seven predicted non-functional variants, thereby validating their predicted effects on splicing in all cases. Conclusions: We demonstrated the accuracy and efficiency of in silico prediction in combination with the cell culture-based full-length gene assay for the classification of SPINK1 intronic variants. Based upon these findings, we propose an operational pipeline for classifying SPINK1 intronic variants in the clinical diagnostic setting.
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Severe infantile isolated exocrine pancreatic insufficiency caused by the complete functional loss of the SPINK1 gene
Human Mutation, 2017Co-Authors: Théa Venet, Emmanuelle Masson, Cécile Talbotec, Kareen Billiemaz, Renaud Touraine, Claire Gay, Sylvie Destombe, David Cooper, Hugues Patural, Jian-min ChenAbstract:Exocrine pancreatic insufficiency (EPI) is rare in children, with most if not all cases occurring as part of syndromic conditions such as cystic fibrosis and Shwachman-Diamond syndrome. Here we report two cases, both presenting with severe EPI around 5 months of age. Characterized by diffuse pancreatic lipomatosis, they otherwise exhibited no remarkable deficiencies in other organs. Novel non-identical homozygous variants (a deletion removing the entire SPINK1 gene and an insertion of a full-length inverted Alu element into the 3'-untranslated region of the SPINK1 gene) resulting in the complete functional loss of the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) were identified in each patient. Having correlated our findings with current knowledge of SPINK1's role in exocrine pancreas pathophysiology, we propose that complete and partial functional losses of the SPINK1 gene are associated with quite distinct phenotypes, the former causing a new pediatric disease entity of severe infantile isolated EPI.
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signal peptide variants that impair secretion of pancreatic secretory trypsin inhibitor SPINK1 cause autosomal dominant hereditary pancreatitis
Human Mutation, 2007Co-Authors: Orsolya Kiraly, Thomas Wartmann, Jian-min Chen, Arnaud Boulling, Heiko Witt, Cedric Le Marechal, Jonas Rosendahl, Cinzia Battaggia, Miklos SahintothAbstract:Variants of the SPINK1 gene encoding pancreatic secretory trypsin inhibitor have been described in association with chronic pancreatitis (CP). These alterations are believed to cause a loss of function by either impairing the trypsin inhibitory activity or reducing expression. Here we report two novel SPINK1 variants in exon 1 that affect the secretory signal peptide. The disease-associated c.41T>G (p.L14R) alteration was found in two European families with autosomal dominant hereditary pancreatitis, whereas the c.36G>C (p.L12F) variant was identified as a frequent alteration in subjects of African descent. The functional effects of both alterations and the previously reported c.41T>C (p.L14P) variant were characterized by activity assays and Western blots of wild-type and mutant SPINK1 expressed in human embryonic kidney 293T and Chinese hamster ovary cells. Alterations p.L14R and p.L14P destined the inhibitor for rapid intracellular degradation and thereby abolished SPINK1 secretion, whereas the p.L12F variant showed no detrimental effect. The results provide the first clear experimental demonstration that alterations that markedly reduce SPINK1 expression are associated with classic hereditary pancreatitis. Therefore, these variants should be classified as severe and regarded as disease-causing rather than disease-modifiers.
Hannu Koistinen - One of the best experts on this subject based on the ideXlab platform.
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An immunocapture-LC-MS-based assay for serum SPINK1 allows simultaneous quantification and detection of SPINK1 variants.
Analytical and bioanalytical chemistry, 2018Co-Authors: Suvi Ravela, Mykola Domanskyy, Hannu Koistinen, Ulf-håkan Stenman, Jakob Stenman, Leena Valmu, Leena Kylänpää, Outi Lindström, Esa Hämäläinen, Outi ItkonenAbstract:Pancreatic secretory trypsin inhibitor Kazal type 1 (SPINK1) is a 6420 Da peptide produced by the pancreas, but also by several other tissues and many tumors. Some mutations of the SPINK1 gene, like the one causing amino acid change N34S, have been shown to confer susceptibility to recurrent or chronic pancreatitis. Detection of such variants are therefore of clinical utility. So far SPINK1 variants have been determined by DNA techniques. We have developed and validated an immunocapture-liquid chromatography-mass spectrometric (IC-LC-MS) assay for the detection and quantification of serum SPINK1, N34S-SPINK1, and P55S-SPINK1. We compared this method with a time-resolved immunofluorometric assay (TR-IFMA) for serum samples and primer extension analysis of DNA samples. We used serum and DNA samples from patients with acute pancreatitis, renal cell carcinoma, or benign urological conditions. With the help of a zygosity score calculated from the respective peak areas using the formula wild-type (wt) SPINK1/(variant SPINK1 + wt SPINK1), we were able to correctly characterize the heterozygotes and homozygotes from the samples with DNA information. The score was then used to characterize the apparent zygosity of the samples with no DNA characterization. The IC-LC-MS method for SPINK1 was linear over the concentration range 0.5–1000 μg/L. The limit of quantitation (LOQ) was 0.5 μg/L. The IC-LC-MS and the TR-IFMA assays showed good correlation. The median zygosity score was 1.00 (95% CI 0.98–1.01, n = 11), 0.55 (95% CI 0.43–0.61, n = 14), and 0.05 (range 0.04–0.07, n = 3) for individuals found to be wt, heterozygous, and homozygous, respectively, for the N34S-SPINK1 variant by DNA analysis. When DNA samples are not available, this assay facilitates identification of the N34S- and P55S-SPINK1 variants also in archival serum samples.
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MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma.
Molecular oncology, 2017Co-Authors: Kati Räsänen, Hannu Koistinen, Ulf-håkan Stenman, Caj Haglund, Kien Xuan Dang, Harri Mustonen, Susanna Lintula, Jakob StenmanAbstract:The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N = 571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.
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Abstract A42: Interleukin-6 induces secretion of SPINK1 and trypsin in colorectal cancer
Cell-Cell Signaling in the Tumor Microenvironment, 2016Co-Authors: Kati Räsänen, Outi Itkonen, Ulf-håkan Stenman, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Hannu KoistinenAbstract: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 Rasanen, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Outi Itkonen, Ulf-Hakan 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.
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Emerging Roles of SPINK1 in Cancer
Clinical chemistry, 2016Co-Authors: Kati Räsänen, Hannu Koistinen, Outi Itkonen, Ulf-håkan StenmanAbstract:BACKGROUND: Tumor-associated trypsin inhibitor (TATI) was originally isolated from the urine of a patient with ovarian cancer. It was later shown to be produced by many other tumors and several normal tissues. It had earlier been isolated from the pancreas and was hence called pancreatic secretory trypsin inhibitor (PSTI). It belongs to a family of protease inhibitors presently called serine peptidase inhibitor Kazal type (SPINK). In the SPINK family TATI/PSTI is SPINK1, which is the name used in this review. CONTENT: In addition to being a protease inhibitor, SPINK1 also acts as an acute-phase reactant and a growth factor. Furthermore, it has been shown to modulate apoptosis. Overexpression of SPINK1 predicts an unfavorable outcome in several cancers and determination of SPINK1 in serum can be used to identify patients at increased risk of aggressive disease. Thus serum SPINK1 can be used as a prognostic tumor marker. Because SPINK1 acts as a growth factor and an inhibitor of apoptosis in some cancers, it has also been suggested that it can be a therapeutic target in cancer. However, because SPINK1 is the major physiological inhibitor of trypsin, inhibition of SPINK1 may increase the risk of pancreatitis. SUMMARY: Taking into account the many functions of SPINK1, assessing the role of SPINK1 in cancer has several potentially important clinical applications ranging from a biomarker to a potential new target for cancer therapy.
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Interleukin-6 increases expression of serine protease inhibitor Kazal type 1 through STAT3 in colorectal adenocarcinoma.
Molecular carcinogenesis, 2015Co-Authors: Kati Räsänen, Outi Itkonen, Ulf-håkan Stenman, Elina Lehtinen, Kristiina Nokelainen, Laura Hautala, Teijo Kuopio, Hannu KoistinenAbstract:Inflammation promotes colorectal cancer (CRC) tumorigenesis, but the underlying molecular mechanisms are still being uncovered. Proinflammatory cytokine interleukin-6 (IL-6) stimulates survival signaling in CRC; inflammatory signals also regulate production and activity of proteases and their inhibitors. Over-expression of serine protease inhibitor Kazal type 1 (SPINK1) predicts an unfavorable outcome in colon cancer. The SPINK1 gene contains an IL-6 responsive element, suggesting it could act as an acute phase reactant. We assessed the connection between IL-6 and SPINK1, and the function and mechanism of this signaling. Our results show that Colo205 and HT-29 cells express and secrete SPINK1, and both fibroblast-derived and recombinant IL-6 further increased the SPINK1 levels. Concurrently CRC cells augmented the IL-6 production in fibroblasts. In CRC tissues cancer cells were positive for SPINK1, whereas IL-6 was found in stromal cells. In Colo205 cells IL-6 also stimulated the secretion of trypsin-1 and -2, the key targets of SPINK1 protease inhibition, whereas in HT-29 cells trypsin-1 and -2 levels remained constantly low. Functionally, both IL-6 and SPINK1 increased the motility of the CRC cells. Mechanistically, IL-6 activated the canonical STAT3 pathway and inhibition of STAT3 phosphorylation decreased the levels of SPINK1, trypsin-1 and -2. Taken together, our results indicate a novel link between inflammatory signals originating from the tumor microenvironment and increased SPINK1 levels. This finding has potential therapeutic implications for targeted therapy, as it confirms that SPINK1 acts as an acute phase reactant and that it participates in the paracrine crosstalk with the tumor microenvironment of colon cancer. © 2015 Wiley Periodicals, Inc.
David C. Whitcomb - One of the best experts on this subject based on the ideXlab platform.
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SPINK1/PSTI Mutations Are Associated with Tropical Pancreatitis in Bangladesh
Pancreatology, 2012Co-Authors: Livio Rossi, Roland H. Pfützer, Shahana Parvin, Soheli Sattar, A.k. Azad Kahn, David C. WhitcombAbstract:Background/Aims: Tropical pancreatitis (TP) refers to a severe type of idiopathic chronic pancreatitis that develops in children in tropical regions of Africa and southern Asia. Phenotypically TP is subdivided into fibrocalculous pancreatic diabetes (FCPD) and tropical calcific pancreatitis without diabetes mellitus (TCP). Recently an association was identified between idiopathic pancreatitis in the USA and Europe and mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene (previously termed pancreatic secretory trypsin inhibitor, PSTI). Our aim was to determine if either form of TP has a genetic basis. Methods: We studied 8 well-characterized patients from Bangladesh with FCPD, 4 with TCP and 4 controls without pancreatic disease. The entire SPINK1 gene was sequenced in these patients. Results: We detected two disease-associated SPINK1 mutations (N34S/IVS1–37T>C and IVS3 + 2T>C) in 6 of 8 patients from Bangladesh with FCPD but not in 4 patients with TCP (p Conclusions: We conclude that SPINK1 mutations are associated with FCPD in Bangladesh. Since SPINK1 mutations in Europeans and North Americans are associated with idiopathic chronic pancreatitis that is phenotypically different from FCPD, we further conclude that mutated SPINK1 markedly increases the risk of developing a variety of pancreatic diseases possibly through a chronic elevation of active trypsin within the pancreas.
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Pathways to Injury in Chronic Pancreatitis: Decoding the Role of the High-Risk SPINK1 N34S Haplotype Using Meta-Analysis
PloS one, 2008Co-Authors: Elie Aoun, Chung-chou H. Chang, Julia B. Greer, Georgios I. Papachristou, M. Michael Barmada, David C. WhitcombAbstract:Background: The complex interactions between recurrent trypsin-mediated pancreatic injury, alcohol-associated pancreatic injury and SPINK1 polymorphisms in chronic pancreatitis (CP) are undefined. We hypothesize that CP occurs as a result of multiple pathological mechanisms (pathways) that are initiated by different metabolic or environmental factors (etiologies) and may be influenced differentially by downstream genetic risk factors. We tested this hypothesis by evaluating the differences in effect size of the high risk SPINK1 N34S haplotype on CP from multiple etiologies after combining clinical reports of SPINK1 N34S frequency using meta-analysis. Methods and Findings: The Pubmed and the Embase databases were reviewed. We studied 24 reports of SPINK1 N34S in CP (2,421 cases, 4,857 controls) using reported etiological factors as surrogates for pathways and multiple meta-analyses to determine the differential effects of SPINK1 N34S between alcoholic and non-alcoholic etiologies. Using estimates of between-study heterogeneity, we sub-classified our 24 studies into four specific clusters. We found that SPINK1 N34S is strongly associated with CP overall (OR 11.00; 95% CI: 7.59-15.93), but the effect of SPINK1 N34S in alcoholic CP (OR 4.98, 95% CI: 3.16-7.85) was significantly smaller than in idiopathic CP (OR 14.97, 95% C.I. = 9.09-24.67) or tropical CP (OR 19.15, 95% C.I. = 8.83-41.56). Studies analyzing familial CP showed very high heterogeneity suggestive of a complex etiology with an I2 = 80.95%. Conclusion: The small effect of SPINK1 N34S in alcoholic subjects suggests that CP is driven through a different pathway that is largely trypsin-independent. The results also suggest that large effect sizes of SPINK1 N34S in small candidate gene studies in CP may be related to a mixture of multiple etiologic pathways leading to the same clinical endpoint. © 2008 Aoun MD et al.
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SPINK1 Mutations Are Associated with Multiple Phenotypes
Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.], 2001Co-Authors: Roland H. Pfützer, David C. WhitcombAbstract:Mutations in the gene encoding for the pancreatic secretory trypsin inhibitor or serine protease inhibitor, Kazal type I (SPINK1) have been associated with different entities of chronic pancreatitis. While there is no doubt about the involvement of SPINK1 mutations in pancreatic inflammatory disease, much controversy has arisen regarding which alterations are associated with disease and what type of disease model should be applied when the SPINK1 gene is examined. This article presents the existing data on SPINK1 mutations in idiopathic chronic pancreatitis, familial pancreatitis, hereditary pancreatitis and tropical pancreatitis. The possible role of SPINK1 mutations and polymorphisms in pancreatic disease is discussed.
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SPINK1 psti mutations are associated with tropical pancreatitis in bangladesh
Pancreatology, 2001Co-Authors: Livio Rossi, Roland H. Pfützer, Shahana Parvin, Soheli Sattar, A Azad K Kahn, David C. WhitcombAbstract:Background/Aims: Tropical pancreatitis (TP) refers to a severe type of idiopathic chronic pancreatitis that develops in children in tropical regions of Africa and southern Asia. Phenotypically TP is subdivided into fibrocalculous pancreatic diabetes (FCPD) and tropical calcific pancreatitis without diabetes mellitus (TCP). Recently an association was identified between idiopathic pancreatitis in the USA and Europe and mutations in the serine protease inhibitor, Kazal type 1 (SPINK1) gene (previously termed pancreatic secretory trypsin inhibitor, PSTI). Our aim was to determine if either form of TP has a genetic basis. Methods: We studied 8 well-characterized patients from Bangladesh with FCPD, 4 with TCP and 4 controls without pancreatic disease. The entire SPINK1 gene was sequenced in these patients. Results: We detected two disease-associated SPINK1 mutations (N34S/IVS1–37T>C and IVS3 + 2T>C) in 6 of 8 patients from Bangladesh with FCPD but not in 4 patients with TCP (p Conclusions: We conclude that SPINK1 mutations are associated with FCPD in Bangladesh. Since SPINK1 mutations in Europeans and North Americans are associated with idiopathic chronic pancreatitis that is phenotypically different from FCPD, we further conclude that mutated SPINK1 markedly increases the risk of developing a variety of pancreatic diseases possibly through a chronic elevation of active trypsin within the pancreas.
Scott A. Tomlins - One of the best experts on this subject based on the ideXlab platform.
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SPINK1 expression is enriched in African American prostate cancer but is not associated with altered immune infiltration or oncologic outcomes post-prostatectomy
Prostate Cancer and Prostatic Diseases, 2019Co-Authors: Farzana A. Faisal, Scott A. Tomlins, Harsimar B. Kaur, Jeffrey J. Tosoian, Edward M. Schaeffer, Tamara L. LotanAbstract:Background The SPINK1 molecular subtype is more common in African-American (AA) men with prostatic adenocarcinoma (PCa) than European Americans (EA). Studies have suggested that SPINK1 expression is associated with more aggressive disease. However, the size, follow-up, and racial diversity of prior patient cohorts have limited our understanding of SPINK1 expression in AA men. The objective was to determine the associations between SPINK1 subtype, race, and oncologic outcomes after radical prostatectomy (RP). Methods A total of 186 AA and 206 EA men who underwent RP were matched according to pathologic grade. We examined SPINK1 status by immunohistochemistry on tissue microarrays using a genetically validated assay. Cox proportional hazard analyses assessed the association of SPINK1 status with oncologic outcomes in race-specific multivariate models. A second objective was to determine the correlation between CD3/CD8 T cell densities with SPINK1 status and race, using immunostaining and automated image analysis. Results SPINK1-positive subtype was present in 25% (45/186) of AA and 15% (30/206) of EA men ( p = 0.013). There were no differences in pathologic grade, pathologic stage, biochemical recurrence (BCR)-free survival, or metastasis-free survival between SPINK1-positive and SPINK1-negative tumors in the overall cohort or by race. In multivariate analyses, SPINK1 expression was not associated with BCR (AA: HR 0.99, 95% CI 0.56–1.75, p = 0.976; EA: HR 0.88, 95% CI 0.43–1.77, p = 0.720) or metastasis (AA: HR 0.79, 95% CI 0.25–2.49, p = 0.691; EA: HR 1.55, 95% CI 0.58–4.11, p = 0.381) in either AA or EA men. There were no significant differences in surrounding CD3/CD8 lymphocyte densities between SPINK1-positive and SPINK1-negative tumors in either race. Conclusions SPINK1-positive subtype is more prevalent in AA than EA men with PCa. Contrary to previous studies, we found that SPINK1 protein expression was not associated with worse pathologic or oncologic outcomes after RP in either AA men or EA men.
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SPINK1 expression is enriched in African American prostate cancer but is not associated with altered immune infiltration or oncologic outcomes post-prostatectomy.
Prostate cancer and prostatic diseases, 2019Co-Authors: Farzana A. Faisal, Scott A. Tomlins, Harsimar B. Kaur, Jeffrey J. Tosoian, Edward M. Schaeffer, Tamara L. LotanAbstract:The SPINK1 molecular subtype is more common in African-American (AA) men with prostatic adenocarcinoma (PCa) than European Americans (EA). Studies have suggested that SPINK1 expression is associated with more aggressive disease. However, the size, follow-up, and racial diversity of prior patient cohorts have limited our understanding of SPINK1 expression in AA men. The objective was to determine the associations between SPINK1 subtype, race, and oncologic outcomes after radical prostatectomy (RP). A total of 186 AA and 206 EA men who underwent RP were matched according to pathologic grade. We examined SPINK1 status by immunohistochemistry on tissue microarrays using a genetically validated assay. Cox proportional hazard analyses assessed the association of SPINK1 status with oncologic outcomes in race-specific multivariate models. A second objective was to determine the correlation between CD3/CD8 T cell densities with SPINK1 status and race, using immunostaining and automated image analysis. SPINK1-positive subtype was present in 25% (45/186) of AA and 15% (30/206) of EA men (p = 0.013). There were no differences in pathologic grade, pathologic stage, biochemical recurrence (BCR)-free survival, or metastasis-free survival between SPINK1-positive and SPINK1-negative tumors in the overall cohort or by race. In multivariate analyses, SPINK1 expression was not associated with BCR (AA: HR 0.99, 95% CI 0.56–1.75, p = 0.976; EA: HR 0.88, 95% CI 0.43–1.77, p = 0.720) or metastasis (AA: HR 0.79, 95% CI 0.25–2.49, p = 0.691; EA: HR 1.55, 95% CI 0.58–4.11, p = 0.381) in either AA or EA men. There were no significant differences in surrounding CD3/CD8 lymphocyte densities between SPINK1-positive and SPINK1-negative tumors in either race. SPINK1-positive subtype is more prevalent in AA than EA men with PCa. Contrary to previous studies, we found that SPINK1 protein expression was not associated with worse pathologic or oncologic outcomes after RP in either AA men or EA men.
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clonal evaluation of prostate cancer foci in biopsies with discontinuous tumor involvement by dual erg SPINK1 immunohistochemistry
Modern Pathology, 2016Co-Authors: Jacqueline Fontugne, Nallasivam Palanisamy, Rohit Mehra, Mark A. Rubin, Kristina Davis, Aaron M. Udager, Andrew S. Mcdaniel, Javed Siddiqui, Juan Miguel Mosquera, Scott A. TomlinsAbstract:The presence of two or more prostate cancer foci separated by intervening benign tissue in a single core is a well-recognized finding on prostate biopsy. Cancer involvement can be measured by including intervening benign tissue or only including the actual cancer involved area. Importantly, this parameter is a common enrollment criterion for active surveillance protocols. We hypothesized that spatially distinct prostate cancer foci in biopsies may arise from separate clones, impacting cancer involvement assessment. Hence, we used dual ERG/SPINK1 immunohistochemistry to determine the frequency of separate clones-when separate tumor foci showed discordant ERG and/or SPINK1 status-in discontinuously involved prostate biopsy cores from two academic institutions. In our cohort of 97 prostate biopsy cores with spatially discrete tumor foci (from 80 patients), discontinuous cancer involvement including intervening tissue ranged from 20 to 100% and Gleason scores ranged from 6 to 9. Twenty-four (25%) of 97 discontinuously involved cores harbored clonally distinct cancer foci by discordant ERG and/or SPINK1 expression status: 58% (14/24) had one ERG(+) focus, and one ERG(-)/SPINK1(-) focus; 29% (7/24) had one SPINK1(+) focus and one ERG(-)/SPINK1(-) focus; and 13% (3/24) had one ERG(+) focus and one SPINK1(+) focus. ERG and SPINK1 overexpression were mutually exclusive in all tumor foci. In summary, our results show that ~25% of discontinuously involved prostate biopsy cores showed tumor foci with discordant ERG/SPINK1 status, consistent with multiclonal disease. The relatively frequent presence of multiclonality in discontinuously involved prostate biopsy cores warrants studies on the potential clinical impact of clonality assessment, particularly in cases where tumor volume in a discontinuous core may impact active surveillance eligibility.
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Clonal evaluation of prostate cancer foci in biopsies with discontinuous tumor involvement by dual ERG/SPINK1 immunohistochemistry
Modern pathology : an official journal of the United States and Canadian Academy of Pathology Inc, 2016Co-Authors: Jacqueline Fontugne, Nallasivam Palanisamy, Rohit Mehra, Mark A. Rubin, Kristina Davis, Aaron M. Udager, Andrew S. Mcdaniel, Javed Siddiqui, Juan Miguel Mosquera, Scott A. TomlinsAbstract:The presence of two or more prostate cancer foci separated by intervening benign tissue in a single core is a well-recognized finding on prostate biopsy. Cancer involvement can be measured by including intervening benign tissue or only including the actual cancer involved area. Importantly, this parameter is a common enrollment criterion for active surveillance protocols. We hypothesized that spatially distinct prostate cancer foci in biopsies may arise from separate clones, impacting cancer involvement assessment. Hence, we used dual ERG/SPINK1 immunohistochemistry to determine the frequency of separate clones-when separate tumor foci showed discordant ERG and/or SPINK1 status-in discontinuously involved prostate biopsy cores from two academic institutions. In our cohort of 97 prostate biopsy cores with spatially discrete tumor foci (from 80 patients), discontinuous cancer involvement including intervening tissue ranged from 20 to 100% and Gleason scores ranged from 6 to 9. Twenty-four (25%) of 97 discontinuously involved cores harbored clonally distinct cancer foci by discordant ERG and/or SPINK1 expression status: 58% (14/24) had one ERG(+) focus, and one ERG(-)/SPINK1(-) focus; 29% (7/24) had one SPINK1(+) focus and one ERG(-)/SPINK1(-) focus; and 13% (3/24) had one ERG(+) focus and one SPINK1(+) focus. ERG and SPINK1 overexpression were mutually exclusive in all tumor foci. In summary, our results show that ~25% of discontinuously involved prostate biopsy cores showed tumor foci with discordant ERG/SPINK1 status, consistent with multiclonal disease. The relatively frequent presence of multiclonality in discontinuously involved prostate biopsy cores warrants studies on the potential clinical impact of clonality assessment, particularly in cases where tumor volume in a discontinuous core may impact active surveillance eligibility.
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novel dual color immunohistochemical methods for detecting erg pten and erg SPINK1 status in prostate carcinoma
Modern Pathology, 2013Co-Authors: Ritu Bhalla, Scott A. Tomlins, Shannon Carskadon, Kyung Park, Javed Siddiqui, Juan Miguel Mosquera, Lakshmi P Kunju, Kelly Christopherson, Connie Cortez, Gary PestanoAbstract:Identification of new molecular markers has led to the molecular classification of prostate cancer based on driving genetic lesions. The translation of these discoveries for clinical use necessitates the development of simple, reliable and rapid detection systems to screen patients for specific molecular aberrations. We developed two dual-color immunohistochemistry-based assays for the simultaneous assessment of ERG–PTEN and ERG–SPINK1 in prostate cancer. A total of 232 cases from 184 localized and 48 metastatic prostate cancers were evaluated for ERG–PTEN and 284 cases from 228 localized and 56 metastatic prostate cancers were evaluated for ERG–SPINK1. Of the 232 cases evaluated for ERG–PTEN, 81 (35%) ERG-positive and 77 (33%) PTEN-deleted cases were identified. Of the 81 ERG-positive cases, PTEN loss was confirmed in 35 (15%) cases by fluorescence in situ hybridization (FISH). PTEN status was concordant in 203 cases (sensitivity 90% and specificity 87%; P<0.0001) by both immunohistochemisty and FISH; however, immunohistochemisty could not distinguish between heterozygous and homozygous deletion status of PTEN. Of the 284 cases evaluated for ERG–SPINK1, 111 (39%) cases were positive for ERG. In the remaining 173 ERG-negative cases, SPINK1 was positive in 26 (9%) cases. SPINK1 expression was found to be mutually exclusive with ERG expression; however, we identified two cases, of which one showed concomitant expression of ERG and SPINK1 in the same tumor foci, and in the second case ERG and SPINK1 were seen in two independent foci of the same tumor nodule. Unlike the homogenous ERG staining in cancer tissues, heterogeneous SPINK1 staining was observed in the majority of the cases. Further studies are required to understand the molecular heterogeneity of cases with concomitant ERG–SPINK1 expression. Automated dual ERG–PTEN and ERG–SPINK1 immunohistochemisty assays are simple, reliable and portable across study sites for the simultaneous assessment of these proteins in prostate cancer.