Serine Peptidase Inhibitor, Kazal Type 1 Research Articles

Serine protease inhibitor Kazal type 1 (SPINK1) promotes proliferation, migration, invasion and radiation resistance in rectal cancer patients receiving concurrent chemoradiotherapy: a potential target for precision medicine

Serine peptidase inhibitor Kazal type-1 (SPINK1), a trypsin kinase inhibitor, is known to be associated with inflammation and pathogenesis. The aim in this study was to demonstrate the clinicopathological role and progression of SPINK1 in rectal cancer (RC) patients undergoing concurrent chemoradiotherapy (CCRT). Immunohistochemical staining for SPINK1 protein expression in 111 RC cases revealed high SPINK1 expression was significantly associated with perineural invasion and poor CCRT response in pre-CCRT specimens. In addition, multivariable analyses showed that pre-CCRT SPINK1 expression was a significant prognostic marker of both overall and disease-free survival in RC patients receiving pre-operative CCRT; furthermore, in vitro studies demonstrated SPINK1 interacted with EGFR to promote the abilities of proliferation, migration and invasion attenuated by SPINK1 si-RNA via ERK, p38, and JNK pathways. SPINK1 was also found to regulate radio-resistance in CRC cell lines. In conclusion, SPINK1 expression is an independent prognostic marker in patients receiving pre-operative CCRT, and SPINK1 regulates proliferation, migration and invasion via EGFR-downstream ERK, p38 and JNK pathways. The phenotypes of radiosensitivity that could be reversed with attenuation of SPINK1 levels suggest that targeting SPINK1 might offer a strategy for optimal precision medicine.

Collision tumors revealed by prospectively assessing subtype-defining molecular alterations in 904 individual prostate cancer foci

BACKGROUNDProstate cancer is multifocal with distinct molecular subtypes. The utility of genomic subtyping has been challenged due to inter- and intrafocal heterogeneity. We sought to characterize the subtype-defining molecular alterations of primary prostate cancer across all tumor foci within radical prostatectomy (RP) specimens and determine the prevalence of collision tumors.METHODSFrom the Early Detection Research Network cohort, we identified 333 prospectively collected RPs from 2010 to 2014 and assessed ETS-related gene (ERG), serine peptidase inhibitor Kazal type 1 (SPINK1), phosphatase and tensin homolog (PTEN), and speckle type BTB/POZ protein (SPOP) molecular status. We utilized dual ERG/SPINK1 immunohistochemistry and fluorescence in situ hybridization to confirm ERG rearrangements and characterize PTEN deletion, as well as high-resolution melting curve analysis and Sanger sequencing to determine SPOP mutation status.RESULTSBased on index focus alone, ERG, SPINK1, PTEN, and SPOP alterations were identified in 47.5%, 10.8%, 14.3%, and 5.1% of RP specimens, respectively. In 233 multifocal RPs with ERG/SPINK1 status in all foci, 139 (59.7%) had discordant molecular alterations between foci. Collision tumors, as defined by discrepant ERG/SPINK1 status within a single focus, were identified in 29 (9.4%) RP specimens.CONCLUSIONInterfocal molecular heterogeneity was identified in about 60% of multifocal RP specimens, and collision tumors were present in about 10%. We present this phenomenon as a model for the intrafocal heterogeneity observed in previous studies and propose that future genomic studies screen for collision tumors to better characterize molecular heterogeneity.FUNDINGEarly Detection Research Network US National Cancer Institute (NCI) 5U01 CA111275-09, Center for Translational Pathology at Weill Cornell Medicine (WCM) Department of Pathology and Laboratory Medicine, US NCI (WCM SPORE in Prostate Cancer, P50CA211024-01), R37CA215040, Damon Runyon Cancer Research Foundation, US MetLife Foundation Family Clinical Investigator Award, Norwegian Cancer Society (grant 208197), and South-Eastern Norway Regional Health Authority (grant 2019016 and 2020063).

A Combined CRISP3 and SPINK1 Prognostic Grade in EPS-Urine and Establishment of Models to Predict Prognosis of Patients With Prostate Cancer.

BackgroundProstate cancer (PCa) is characterized by significant heterogeneity. Thus, novel prognostic indicators are required to improve prognosis and treatment.MethodsCysteine rich secretory protein 3 (CRISP3) and serine peptidase inhibitor Kazal type 1 (SPINK1) levels in expressed prostatic secretion (EPS)-urine collected during digital rectal examination of 496 patients histologically diagnosed with PCa were detected via enzyme-linked immunosorbent assay. A combined CRISP3 and SPINK1 prognostic grade (CSPG) was defined using cut-off values from receiver operating characteristic curves. Log-rank Kaplan-Meier survival curves investigated differences in prognosis between groups. Univariate and multivariate Cox analyses investigated the CSPG relationship with biochemical recurrence (BCR), cancer-specific survival (CSS), and overall survival (OS). Three prognostic models were developed and validated.ConclusionsCRISP3 and SPINK1 levels increased with Gleason score progression, pathological T stage, and metastasis status. CSPG in EPS-urine, which was an effective independent prognostic variable, accurately predicted the prognosis of patients with PCa. Three clinical prognostic models using the CSPG for BCR, CSS, and OS were developed and validated.

A novel gene signature for prognosis prediction and chemotherapy response in patients with pancreatic cancer.

Pancreatic cancer is a lethal disease. Chemoresistance is one of the characteristics of pancreatic cancer and leads to a poor prognosis. This study built an effective predictive model for personalized treatment and explored the molecular mechanism of chemoresistance. A four-gene signature, including serine peptidase inhibitor Kazal type 1 (SPINK1), anoctamin 1 (ANO1), desmoglein 3 (DSG3) and GTPase, IMAP family member 1 (GIMAP1) was identified and associated with prognosis and chemoresistance in the training group. An internal testing dataset and the external dataset, GSE57495, were used for validation and showed a good performance of the gene signature. The high-risk group was enriched with multiple oncological pathways related to immunosuppression and was correlated with epidermal growth factor receptor (EGFR) expression, a target molecule of Erlotinib. In conclusion, this study identified a four-gene signature and established two nomograms for predicting prognosis and chemotherapy responses in patients with pancreatic cancer. The clinical value of the nomogram was evaluated by decision curve analysis (DCA). It showed that these may be helpful for clinical treatment decision-making and the discovery of the potential molecular mechanism and therapy targets for pancreatic cancer.

Functional Roles of SPINK1 in Cancers.

Serine Peptidase Inhibitor Kazal Type 1 (SPINK1) is a secreted protein known as a protease inhibitor of trypsin in the pancreas. However, emerging evidence shows its function in promoting cancer progression in various types of cancer. SPINK1 modulated tumor malignancies and induced the activation of the downstream signaling of epidermal growth factor receptor (EGFR) in cancer cells, due to the structural similarity with epidermal growth factor (EGF). The discoverable SPINK1 somatic mutations, expressional signatures, and prognostic significances in various types of cancer have attracted attention as a cancer biomarker in clinical applications. Emerging findings further clarify the direct and indirect biological effects of SPINK1 in regulating cancer proliferation, metastasis, drug resistance, transdifferentiation, and cancer stemness, warranting the exploration of the SPINK1-mediated molecular mechanism to identify a therapeutic strategy. In this review article, we first integrate the transcriptomic data of different types of cancer with clinical information and recent findings of SPINK1-mediated malignant phenotypes. In addition, a comprehensive summary of SPINK1 expression in a pan-cancer panel and individual cell types of specific organs at the single-cell level is presented to indicate the potential sites of tumorigenesis, which has not yet been reported. This review aims to shed light on the roles of SPINK1 in cancer and provide guidance and potential directions for scientists in this field.

Transition zone prostate cancer is associated with better clinical outcomes than peripheral zone cancer.

ObjectivesTo determine the biological significance of zonal origins in prostate cancer.Patients and methodsAltogether, 270 consecutive radical prostatectomy cases from 2009 to 2012 were adopted. Cases were divided into those having transition zone (TZ) cancer or peripheral zone (PZ) cancer. Cases with indeterminate tumor location and central zone cancers were excluded from the analyses. Prognosis and clinicopathological features were compared between the two tumor locations. Biochemical recurrence (BCR) and clinical progression (CP) were adopted as prognostic outcome measures. Immunohistochemical features of the v‐ets avian erythroblastosis virus E26 oncogene homolog (ERG)/serine peptidase inhibitor, Kazal‐type 1 (SPINK1) status, and loss of phosphatase and tensin homolog (PTEN‐loss), as well as conventional preoperative and postoperative characteristics, were analyzed.ResultsThis cohort comprised 93 cases of TZ cancer and 160 cases of PZ cancer. TZ cancer cases showed significantly higher BCR and CP‐free survival rate than PZ cancer cases. Notably, no TZ cancer cases developed CP during the 7.8 years of median follow‐up time. Tumor location was an independent predictive factor for BCR in the multivariate analysis. Additionally, TZ cancer cases showed a significantly lower prevalence of ERG‐overexpression and PTEN‐loss than PZ cancer cases (3.2% vs 20.1% and 2.2% vs 18.2%, respectively).ConclusionTZ cancer cases showed a better prognosis and different immunohistochemical features. Conservative treatment strategies could be considered for TZ cancer cases.

Dramatic increase in serum trypsinogens, SPINK1 and hCGβ in aortic surgery patients after hypothermic circulatory arrest

The concentrations of several diagnostic markers have been found to increase dramatically in critically ill patients with a severe disturbance of normal physiological homeostasis, without indication of the diseases they are normally associated with. To prevent false diagnoses and inappropriate treatments of critically ill patients, it is important that the markers aiding the selection of second-line treatments are evaluated in such patients and not only in the healthy population and patients with diseases the markers are associated with. The levels of trypsinogen isoenzymes, the trypsin inhibitor serine peptidase inhibitor Kazal type 1 (SPINK1), hCG and hCGβ, which are used as pancreatitis and cancer markers, were analyzed by immunoassays from serum samples of 17 adult patients who have undergone surgery of the ascending aorta during hypothermic circulatory arrest (HCA) with optional selective cerebral perfusion. Highly elevated levels of trypsinogen-1, -2 and -3, SPINK1 and hCGβ were observed in patients after HCA. This was accompanied by increased concentrations of S100β and NSE. In conclusion, this study highlights the importance of critically evaluating the markers used for aiding selection of second line of treatments in critically ill patients.

Abstract C128: Reprogramming transcription factors SOX2 and REST modulates SPINK1 expression in governing cellular plasticity in prostate cancer

Abstract Androgen deprivation therapy (ADT) remains the clinical paradigm for the management of prostate cancer (PCa) patients. However, most of the cases inevitably become resistant to ADT, leading to a more aggressive, hormone-refractory stage known as castrate-resistant prostate cancer (CRPC). A subset of CRPC patients undergoing ADT develop tumors with low AR-signaling dependence where the luminal prostate cancer cells consequently acquire alternative lineage programs, resulting in the development of neuroendocrine prostate cancer (NEPC). In the past decade, several molecular features have been associated with progression of the adenocarcinoma to NEPC, including loss of RE1-silencing transcription factor (REST), N-MYC proto-oncogene amplification, upregulation of Aurora kinase A, neural transcription factor BRN2, and reprogramming factor SRY (sex determining region Y)-box 2 (SOX2). Nonetheless, the underlying molecular mechanism involved in lineage plasticity in neuroendocrine (NE) transdifferentaition is poorly understood and remains a matter of speculation. Overexpression of Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) represents the second major molecular PCa subtype (~15-20% of the cases) associated with aggressive stage and poor patient outcome. Oncogenic role of SPINK1 in PCa has already been established, and recently SPINK1 produced in the tumor stroma was found to act as a senescence-associated secretory factor and contributes to chemoresistance in a paracrine manner. Here, we show that androgen receptor (AR) along with its corepressor REST functions as a direct transcriptional repressor of SPINK1, and blocking AR signaling using anti-androgens relieve this repression, leading to increase in SPINK1 expression. We also show that the lineage reprogramming factor SOX2 binds to the SPINK1 promoter and positively regulate its expression in androgen deprivation induced NE-transdifferentiated PCa cells. Conversely, silencing SPINK1 in the NE-transdifferentiated PCa cells result in reduced expression of epithelial-mesenchymal transition (EMT) and neuroendocrine markers accompanied with a concomitant decrease in the neurite-like projections. Moreover, castration-resistant mice xenografts treated with anti-androgens show increase in SPINK1 levels as well as EMT and neuroendocrine markers. Likewise, higher SPINK1 expression was observed in NE patient-derived organoids and NEPC clinical specimens, indicating its plausible role in cellular plasticity and NE progression. Since, REST along with AR negatively regulates SPINK1 expression, and Casein kinase 1 elicits ubiquitin-mediated proteasomal degradation of REST. Therefore, we treated SPINK1-positive PCa cells with Casein kinase 1 inhibitor (iCK1), which results in restoration of the REST expression, leading to transcriptional repression of SPINK1 as well as decrease in SPINK1-mediated oncogenesis. Taken together, we highlight the clinical complications associated with high SPINK1 levels and discovered its possible role in maintaining cellular plasticity in prostate cancer cells. Notably, stabilization of REST levels using iCK1 suggests a novel therapeutic strategy for the management of SPINK1-positive subtype and also open new avenues for the treatment modalities for CRPC patients. Citation Format: Nishat Manzar, Ritika Tiwari, Vipul Bhatia, Anjali Yadav, Shannon Carskadon, Nilesh Gupta, Amina Zoubeidi, Matti Poutanen, Himisha Beltran, Nallasivam Palanisamy, Bushra Ateeq. Reprogramming transcription factors SOX2 and REST modulates SPINK1 expression in governing cellular plasticity in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C128. doi:10.1158/1535-7163.TARG-19-C128

Dynamics of Genomic, Epigenomic, and Transcriptomic Aberrations during Stepwise Hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) undergoes a stepwise progression from liver cirrhosis to low-grade dysplastic nodule (LGDN), high-grade dysplastic nodule (HGDN), early HCC (eHCC), and progressed HCC (pHCC). Here, we profiled multilayered genomic, epigenomic, and transcriptomic aberrations in the stepwise hepatocarcinogenesis. Initial DNA methylation was observed in eHCC (e.g., DKK3, SALL3, and SOX1) while more extensive methylation was observed in pHCC. In addition, eHCCs showed an initial loss of DNA copy numbers of tumor suppressor genes in the 4q and 13q regions, thereby conferring survival benefits to cancer cells. Transcriptome analysis revealed that HGDNs expressed endoplasmic reticulum (ER) stress-related genes, while eHCC started to express oncogenes. Furthermore, integrative analysis indicated that expression of the serine peptidase inhibitor, Kazal type 1 (SPINK1), played a pivotal role in eHCC development. Significant demethylation of SPINK1 was observed in eHCC compared to HGDN. The study also demonstrated that ER stress may induce SPINK1 demethylation and expression in liver cancer cells. In conclusion, these results reveal the dynamics of multiomic aberrations during malignant conversion of liver cancer, thus providing novel pathobiological insights into hepatocarcinogenesis. SIGNIFICANCE: Multiomics profiling and integrative analyses of stepwise hepatocarcinogenesis reveal novel mechanistic and clinical insights into hepatocarcinogenesis.

Abstract 5225: Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer

Abstract The Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) overexpression represents the second-largest prostate cancer (PCa) subtype associated with increased risk of recurrence and poor prognosis. Regardless of molecular subtype, androgen-deprivation therapy (ADT) remains the mainstay treatment for locally advanced and metastatic PCa patients. However, majority of the treated individuals eventually progress to castration-resistant stage and a subset of these patients develop ADT-induced neuroendocrine prostate cancer. Despite evidences of detrimental effects of ADT on PCa, possible role of androgen signaling in SPINK1-mediated prostate oncogenesis remains unexplored. Here, we show that androgen receptor (AR) functions as a direct transcriptional repressor of SPINK1, and blocking AR signaling relieves its repression, leading to upregulation of SPINK1. In agreement, we observe an inverse association between SPINK1 levels and AR expression across multiple patient cohorts, and in neuroendocrine differentiated LNCaP cells. We show that AR and its corepressor, the RE1-silencing transcription factor (REST), occupy SPINK1 promoter and inhibits its transcription. On the other hand, in the absence of AR, lineage reprogramming factor SOX2 in turn binds to SPINK1 promoter leading to its positive transcriptional regulation in androgen-deprived conditions with concomitant increase in neuroendocrine markers. Additionally, stable knockdown of SPINK1 results in reduced epithelial-mesenchymal transition, decreased stemness and drug resistance. Collectively, our findings provide a plausible explanation to the paradoxical clinical outcomes of ADT, arising due to increase in SPINK1 levels. Finally, we emphasize the need to take a well-informed decision prior to ADT and develop alternative therapeutic strategies for castrate-resistant PCa patients. Citation Format: Ritika Tiwari, Nishat Manzar, Vipul Bhatia, Anjali Yadav, Shannon Carskadon, Nilesh Gupta, Amina Zoubeidi, Nallasivam Palanisamy, Bushra Ateeq. Androgen deprivation upregulates SPINK1 expression and potentiates cellular plasticity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5225.

Epigenetic Silencing of miRNA-338-5p and miRNA-421 Drives SPINK1-Positive Prostate Cancer.

Serine peptidase inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in prostate cancer remains largely unknown. miRNA prediction tools were employed to examine the SPINK1-3'UTR for miRNA binding. Luciferase reporter assays were performed to confirm the SPINK1-3'UTR binding of shortlisted miR-338-5p/miR-421. Furthermore, miR-338-5p/-421-overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and a mouse xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. IHC and RNA in situ hybridization were carried out on prostate cancer patients' tissue microarray for SPINK1 and EZH2 expression, respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421-regulatory regions. Bisulfite sequencing and methylated DNA immunoprecipitation were performed on prostate cancer cell lines and patients' specimens. We established a critical role of miRNA-338-5p/-421 in posttranscriptional regulation of SPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive cells abrogates oncogenic properties including cell-cycle progression, stemness, and drug resistance, and shows reduced tumor burden and distant metastases in a mouse model. Importantly, we show that patients with SPINK1-positive prostate cancer exhibit increased EZH2 expression, suggesting its role in epigenetic silencing of miRNA-338-5p/-421. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive prostate cancer cells and patients' specimens confirms epigenetic silencing. Our findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive prostate cancer, although restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.See related commentary by Bjartell, p. 2679.

Serum Serine Peptidase Inhibitor Kazal-Type 1, Trypsinogens 1 to 3, and Complex of Trypsin 2 and α1-Antitrypsin in the Diagnosis of Severe Acute Pancreatitis.

We explored prediction of severe acute pancreatitis (AP) and development of organ dysfunction (OD). Serum concentrations of serine peptidase inhibitor Kazal type 1 (SPINK1), trypsinogen 1, trypsinogen 2, and trypsinogen 3, complex between trypsin 2 and α1-antitrypsin, serum C-reactive protein, creatinine, and pancreatic amylase were measured in 239 AP patients with disease onset within 72 hours. SPINK1 distinguished most accurately patients who later developed severe AP. The area under the receiver operating characteristic curve for SPINK1 was 0.742, followed by trypsinogen 2 (0.726), complex between trypsin 2 and α1-antitrypsin (0.657), creatinine (0.656), trypsinogen 1 (0.652), trypsinogen 3 (0.557), and C-reactive protein (0.499). With a cutoff of 166 μg/L, SPINK1 had a specificity of 93%, a sensitivity of 48%, and diagnostic odds ratio of 11.52. In multivariate logistic regression analysis, only SPINK1 was an independent predictor of severe AP among patients presenting without OD on admission (P < 0.001). Plasma levels of the biomarkers and creatinine correlated with the severity of AP and development of OD. In patients presenting without OD at admission, SPINK1 was an independent marker for later development of severe AP.

SPINK-1 Polymorphism as a Pancreatitis Risk Factor

Pancreatitis in both acute and chronic variants is a common health concern in the US as well as globally. While the most common etiologies for disease remain gallstone impaction in the common bile duct and alcohol abuse, recent studies have shown that genetics may play a significant role as well. Unfortunately, this correlation is not clearly defined and at present, we lack the ability to identify which patients with known pancreatic genetic polymorphisms will develop pancreatitis. We describe the case of a middle-aged male who presented with recurrent pancreatitis in the setting of the serine peptidase inhibitor, Kazal type 1 (SPINK-1) genetic polymorphism. Additionally, we discuss the mechanism behind SPINK-1 polymorphisms in the development of pancreatitis as well as the role of genetic screening for the polymorphism in the general population.

Abstract A060: Repression of microRNA-338/421 by EZH2 represents a novel mechanism for SPINK1-positive prostate cancers

Abstract Prostate cancer (PCa) is a clinically heterogeneous disease marked by variability in patient prognosis. The intratumoral and interpatient heterogeneity formed the basis of molecular stratification of the disease. Overexpression of Serine Peptidase Inhibitor, Kazal type-1 (SPINK1), was found to be the second most recurrent (~10-15%) PCa subtype, after highly recurrent (~50%) androgen-driven TMPRSS2-ERG genetic rearrangement. Unlike amplification of ERBB2 in breast cancer, SPINK1 overexpression has not been associated with gene amplification or rearrangement. Nonetheless, molecular mechanism underlying its upregulation in cancer is poorly understood and remains a matter of conjecture. Using in silico microRNA prediction tools, we shortlisted three miRNAs, of which miR-338-5p and miR-421 revealed an inverse correlation with SPINK1 in The Cancer Genome Atlas (TCGA) prostate adenocarcinoma RNA-Seq dataset (n=106), which was validated in our PCa specimens (n=20). Here, we showed that miR-338-5p and miR-421 post-transcriptionally regulate SPINK1 by binding to its 3′ UTR. We established that miR-338-5p and miR-421 mediate several cellular responses against SPINK1-positive cancer by inducing S-phase cell-cycle arrest, inhibiting epithelial-to-mesenchymal transition (EMT), cancer stemness, and drug resistance. Moreover, ectopic expression of miR-338-5p and miR-421 abrogates SPINK1-mediated oncogenesis, tumor growth, and distant metastases in murine xenograft model. Further, mechanistically we demonstrate that Polycomb group protein enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing by establishing histone H3K27me3 repressive marks and DNA methylation, 5’ methyl cytosine (5mC) marks on the promoters of miR-338-5p and miR-421 in SPINK1-positive subtype. Thus, restoring miR-338-5p and miR-421 expression using either epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis by targeting multiple oncogenic pathways and eliciting anticancer pleiotropic effects. Taken together, the present study for the first time unravels the molecular mechanism underlying SPINK1 overexpression and thereby opens up new avenues for the use of miR-338-5p and miR-421 replacement therapy for the treatment of SPINK1-positive malignancies. Citation Format: Vipul Bhatia, Anjali Yadav, Ritika Tiwari, Shivansh Nigam, Apul Goel, Bushra Ateeq. Repression of microRNA-338/421 by EZH2 represents a novel mechanism for SPINK1-positive prostate cancers [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A060.

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.

MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma.

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.

Transcriptomic features of primary prostate cancer and their prognostic relevance to castration-resistant prostate cancer.

Although various mechanisms of castration-resistant prostate cancer (CRPC) have been discovered, reliable biomarkers for monitoring CRPC progression are lacking. We sought to identify molecules that predict the progression of advanced prostate cancer (AdvPC) into CRPC. The study used primary-site samples (N=45 for next-generation sequencing (NGS); N=243 for real-time polymerase chain reaction) from patients with prostate cancer (PC). Five public databases containing microarray data of AdvPC and CRPC samples were analyzed. The NGS data showed that each progression step in PC associated with distinct gene expression profiles. Androgen receptor (AR) associated with tumorigenesis, advanced progression, and progression into CRPC. Analysis of the paired and unpaired AdvPC and CRPC samples in the NGS cohort showed that 15 genes associated with progression into CRPC. This was validated by cohort-1 and public database analyses. Analysis of the third cohort with AdvPC showed that higher serine peptidase inhibitor, Kazal type 1 (SPINK1) and lower Sp8 transcription factor (SP8) expression associated with progression into CRPC (log-rank test, both P<0.05). Multivariate regression analysis showed that higher SPINK1 (Hazard Ratio (HR)=4.506, 95% confidence intervals (CI)=1.175–17.29, P=0.028) and lower SP8 (HR=0.199, 95% CI=0.063–0.632, P=0.006) expression independently predicted progression into CRPC. Gene network analysis showed that CRPC progression may be mediated through the AR-SPINK1 pathway by a HNF1A-based gene network. Taken together, our results suggest thatSPINK1 and SP8 may be useful for classifying patients with AdvPC who have a higher risk of progressing to CRPC.

Serine peptidase inhibitor Kazal type 1 (SPINK1) as novel downstream effector of the cadherin-17/β-catenin axis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide. Previously, we reported that cadherin-17 (CDH17) and its related CDH17/β-catenin axis may be responsible for inducing HCC in a subset of patients exhibiting CDH17 over-expression. Here we aimed at obtaining a better understanding of the CDH17-related HCC biology and to obtain further indications for the design of targeted therapies in CDH17 over-expressing HCC patients. We found that SPINK1 acts as a downstream effector of the CDH17/β-catenin axis in HCC. In addition, we found that SPINK1 expression exhibited a positive correlation with CDH17 expression in human HCCs and was over-expressed in up to 70% of the tumors. We identified SPINK1 as a downstream effector of the CDH17/β-catenin axis using a spectrum of in vitro assays, including gene expression modulation and inhibitor assays, bioinformatics analyses and luciferase reporter assays. These in vitro results were validated in primary human HCCs, including the observation that alteration in β-catenin expression (a core component of the CDH17/β-catenin axis) in tumors affects SPINK1 serum levels in HCC patients. Similar to CDH17, SPINK1 expression in HCC cells was found to be associated with specific tumor-related properties via activating the c-Raf/MEK/ERK pathway. Our current data substantiate our knowledge on the role of CDH17 in the biology of HCC and suggest that components of the CDH17/β-catenin axis may serve as therapeutic targets in CDH17 over-expressing HCC patients.

The association between SPINK1 and clinical outcomes in patients with prostate cancer: a systematic review and meta-analysis.

Evidence of the prognostic role of serine peptidase inhibitor Kazal type 1 (SPINK1) in prostate cancer (PCa) is controversial. The aim of this study was, therefore, to evaluate the association between SPINK1 and clinical outcomes in PCa. Searches were made of PubMed, Medline, Embase, and the China Biology Medicine disc (CBMdisc) up to January 2017. The Newcastle–Ottawa Scale was used to assess the risk of bias of included studies. RevMan software was used to perform meta-analysis, and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) method was employed for assessing the quality of the evidence. Ten studies with 17,161 patients were included in the analysis. Random-effect models were adopted for all outcomes with significant heterogeneities. In patients treated with radical prostatectomy, SPINK1 was associated with biochemical recurrence (BCR) (hazard ratio [HR] =1.41, 95% confidence interval [CI]: 1.01–1.97; P=0.04), but not PCa-specific mortality (HR =0.93, 95% CI: 0.33–2.57; P=0.88), and overall survival (OS) (HR =0.89, 95% CI: 0.58–1.35; P=0.57). In metastatic PCa, SPINK1 was significantly associated with castration-resistant PCa-free survival (HR =3.87, 95% CI: 1.87–8.00; P=0.0003) and OS (HR =2.59, 95% CI: 1.16–5.78; P=0.02). However, the quality of the evidence was very low for all study outcome measures. In conclusion, although SPINK1 was not a predictor of PCa mortality or OS among patients who underwent radical prostatectomy, it may have prognostic value in metastatic PCa.

Frequency of Tabagism and N34S and P55S Mutations of Serine Peptidase Inhibitor, Kazal Type 1 (SPINK1) and R254W Mutation of Chymotrypsin C (CTRC) in Patients With Chronic Pancreatitis and Controls.

This study aimed to investigate the association between chronic pancreatitis and smoking or genetic mutations. The study sample comprised 148 patients with chronic pancreatitis, 110 chronic alcoholic subjects without pancreatic disease, and 297 volunteer blood donors. Of the patients with chronic pancreatitis, 74% had alcoholic etiology and 26% had idiopathic pancreatitis. The frequency of smoking was 91.4% in patients with alcoholic pancreatitis, higher than 73.3% in alcoholic subjects without pancreatitis (P < 0.01). The difference in smoking frequency was not significant between the patients with idiopathic pancreatitis and blood donors. The N34S mutation of serine peptidase inhibitor, Kazal type 1 (SPINK1) was found in 2.7% of patients with chronic alcoholic pancreatitis, in 5.3% of patients with idiopathic pancreatitis, and in 0.4% of blood donors (P = 0.02). The P55S mutation of SPINK1 was found in 2.7% of patients with alcoholic pancreatitis and in 0.7% of blood donors (P = 0.12). The R254W mutation of chymotrypsin C was found in 0.9% of patients with alcoholic pancreatitis, in 0.9% of chronic alcoholic subjects without pancreatitis, and in 0.4% of blood donors (P = 0.75). In all cases, the mutations were heterozygous. Smoking and the N34S mutation of SPINK1 were positively correlated with chronic pancreatitis.