SPINK1 Expression Research Articles

Abstract 712: Antitumor activity of SPINK1 monoclonal antibody in a subset of SPINK1 positive ETS-negative prostate cancer

Abstract We have identified SPINK1 outlier expression in an aggressive subset of TMPRSS2:ETS-negative prostate cancers (10% of all prostate cancers) by outlier meta-analysis (meta-COPA). We have further validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier expression can be detected non-invasively in urine, and is a useful prognostic marker in prostate cancer (Tomlins et al., 2008 and Laxman et al., 2008). Here we examined the in vitro and in vivo effects of SPINK1 outlier expression. We demonstrate that exogenous SPINK1 recombinant protein stimulate cell proliferation in benign and cancerous prostate cells. Benign prostate epithelial cells RWPE treated with SPINK1 recombinant protein or conditioned medium (CM) from 22RV1 cells (SPINK1+) showed dramatic increase in cell invasion in Boyden chamber Matrigel invasion assay, which was attenuated in the presence of an anti-SPINK1 monoclonal antibody (mAb). We also showed that invasion of 22RV1 cells is attenuated with SPINK1 mAb, while SPINK1 mAb has no effect on the invasion of PC3 cells (SPINK1−) invasion. SiRNA knockdown of SPINK1 in 22RV1 cells attenuated invasion, which could be rescued by recombinant SPINK1 protein or 22RV1 CM. Stable knockdown of SPINK1 in 22RV1 cells using shRNA resulted in decreased cell proliferation, cell invasion and motility. Moreover, stable knockdown of SPINK1 in 22RV1-Luc cells showed reduced tumor burden in an in vivo xenograft model. The preclinical in vivo efficacy of SPINK1 mAb was evaluated in SPINK1 positive 22RV1-Luc xenograft model established in BalbC nu. nu mice. Antitumor activities were determined from the analyses of tumor growth inhibition, defined as the decrease in the mean tumor volume for SPINK1 mAb treated mice versus IgG mAb treated mice. Twice a week intra-peritoneal administration of SPINK1 mAb (10mg/kg) resulted in a 65% reduction of tumor burden. This study provides the rationale for the development of humanized anti-SPINK1 monoclonal antibodies for clinical targeting of the subset of patients with SPINK1 positive and ETS-negative prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 712.

Serine Protease Inhibitor Kazal Type 1 Promotes Proliferation of Pancreatic Cancer Cells through the Epidermal Growth Factor Receptor

Serine protease inhibitor, Kazal type 1 (SPINK1) is expressed not only in normal human pancreatic acinar cells but also in a variety of pancreatic ductal neoplasms. There are structural similarities between SPINK1 and epidermal growth factor (EGF). Hence, we hypothesized that SPINK1 binds to EGF receptor (EGFR) to activate its downstream signaling. We first showed that SPINK1 induced proliferation of NIH 3T3 cells and pancreatic cancer cell lines. We showed that SPINK1 coprecipitated with EGFR in an immunoprecipitation experiment and that the binding affinity of SPINK1 to EGFR was about half of that of EGF using quartz-crystal microbalance (QCM) technique. As expected, EGFR and its downstream molecules, signal transducer and activator of transcription 3, v-Akt murine thymoma viral oncogene homologue, and extracellular signal-regulated kinase 1/2, were phosphorylated by SPINK1 as well as EGF. To determine which pathway is the most important for cell growth, we further analyzed the effect of inhibitors. Growth stimulation by EGF or SPINK1 was completely inhibited by EGFR and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor but not by Janus-activated kinase and phosphoinositide 3-kinase inhibitors. To further analyze the clinical importance of SPINK1 in the development of pancreatic cancer, we examined the expression of SPINK1 and EGFR in pancreatic tubular adenocarcinomas and pancreatic intraepithelial neoplasm. Both SPNK1 and EGFR were coexpressed not only in the early stage of cancer, PanIN-1A, but also in advanced stages. Taken together, these results suggest that SPINK1 stimulates the proliferation of pancreatic cancer cells through the EGFR/mitogen-activated protein kinase cascade.

The Role of SPINK1 in ETS Rearrangement-Negative Prostate Cancers

ETS gene fusions have been characterized in a majority of prostate cancers; however, the key molecular alterations in ETS-negative cancers are unclear. Here we used an outlier meta-analysis (meta-COPA) to identify SPINK1 outlier expression exclusively in a subset of ETS rearrangement-negative cancers ( approximately 10% of total cases). We validated the mutual exclusivity of SPINK1 expression and ETS fusion status, demonstrated that SPINK1 outlier expression can be detected noninvasively in urine, and observed that SPINK1 outlier expression is an independent predictor of biochemical recurrence after resection. We identified the aggressive 22RV1 cell line as a SPINK1 outlier expression model and demonstrate that SPINK1 knockdown in 22RV1 attenuates invasion, suggesting a functional role in ETS rearrangement-negative prostate cancers.

A First-Generation Multiplex Biomarker Analysis of Urine for the Early Detection of Prostate Cancer

Although prostate-specific antigen (PSA) serum level is currently the standard of care for prostate cancer screening in the United States, it lacks ideal specificity and additional biomarkers are needed to supplement or potentially replace serum PSA testing. Emerging evidence suggests that monitoring the noncoding RNA transcript PCA3 in urine may be useful in detecting prostate cancer in patients with elevated PSA levels. Here, we show that a multiplex panel of urine transcripts outperforms PCA3 transcript alone for the detection of prostate cancer. We measured the expression of seven putative prostate cancer biomarkers, including PCA3, in sedimented urine using quantitative PCR on a cohort of 234 patients presenting for biopsy or radical prostatectomy. By univariate analysis, we found that increased GOLPH2, SPINK1, and PCA3 transcript expression and TMPRSS2:ERG fusion status were significant predictors of prostate cancer. Multivariate regression analysis showed that a multiplexed model, including these biomarkers, outperformed serum PSA or PCA3 alone in detecting prostate cancer. The area under the receiver-operating characteristic curve was 0.758 for the multiplexed model versus 0.662 for PCA3 alone (P = 0.003). The sensitivity and specificity for the multiplexed model were 65.9% and 76.0%, respectively, and the positive and negative predictive values were 79.8% and 60.8%, respectively. Taken together, these results provide the framework for the development of highly optimized, multiplex urine biomarker tests for more accurate detection of prostate cancer.

The SPINK gene family and celiac disease susceptibility.

The gene family of serine protease inhibitors of the Kazal type (SPINK) are functional and positional candidate genes for celiac disease (CD). Our aim was to assess the gut mucosal gene expression and genetic association of SPINK1, -2, -4, and -5 in the Dutch CD population. Gene expression was determined for all four SPINK genes by quantitative reverse-transcription polymerase chain reaction in duodenal biopsy samples from untreated (n = 15) and diet-treated patients (n = 31) and controls (n = 16). Genetic association of the four SPINK genes was tested within a total of 18 haplotype tagging SNPs, one coding SNP, 310 patients, and 180 controls. The SPINK4 study cohort was further expanded to include 479 CD cases and 540 controls. SPINK4 DNA sequence analysis was performed on six members of a multigeneration CD family to detect possible point mutations or deletions. SPINK4 showed differential gene expression, which was at its highest in untreated patients and dropped sharply upon commencement of a gluten-free diet. Genetic association tests for all four SPINK genes were negative, including SPINK4 in the extended case/control cohort. No SPINK4 mutations or deletions were observed in the multigeneration CD family with linkage to chromosome 9p21-13 nor was the coding SNP disease-specific. SPINK4 exhibits CD pathology-related differential gene expression, likely derived from altered goblet cell activity. All of the four SPINK genes tested do not contribute to the genetic risk for CD in the Dutch population.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-007-0199-5) contains supplementary material, which is available to authorized users.

Signal peptide variants that impair secretion of pancreatic secretory trypsin inhibitor (SPINK1) cause autosomal dominant hereditary pancreatitis

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.