Association of high miR-27a, miR-206, and miR-214 expression with poor patient prognosis and increased chemoresistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, associated with early metastasis and recurrence as well as poor patient outcome. TNBC does not or weakly respond to hormonal or HER2-targeted therapies. Therefore, there is a strong need to identify other potential molecular targets for TNBC therapy. Micro-RNAs play important roles in the post-transcriptional regulation of gene expression. Thus, micro-RNAs, displaying an association between elevated expression and poor patient prognosis, may represent candidates for such novel tumor targets. In the present study, we evaluated the prognostic impact of miR-27a, miR-206, and miR-214 in TNBC via qPCR in tumor tissue (n=146). In univariate Cox regression analysis, elevated expression of all three analyzed micro-RNAs was significantly associated with shortened disease-free survival (hazard ratio [HR] for miR-27a: 1.85, P=0.038; miR-206: 1.83, P=0.041; miR-214: 2.06, P=0.012). In multivariable analysis, the micro-RNAs remained independent biomarkers for disease-free survival (HR for miR-27a: 1.99, P=0.033; miR-206: 2.14, P=0.018; miR-214: 2.01, P=0.026). Furthermore, our results suggest that elevated levels of these micro-RNAs are linked to enhanced resistance to chemotherapy. Based on the association of high expression levels with shortened patient survival and increased chemoresistance, miR-27a, miR-206, and miR-214 may represent novel molecular targets for TNBC.

tRNA-derived small RNA 3'U-tRFValCAC promotes tumour migration and early progression in ovarian cancer.

INTRODUCTION: Despite recent advances in epithelial ovarian cancer (EOC) management, the highly heterogenous histological/molecular tumour background and patients' treatment response obstructs personalised prognosis and therapeutics. Herein, we have studied the role and clinical utility of the novel subclass of tRNA-derived small RNA fragments emerging via 3'-trailer processing of pre-tRNAs (3'U-tRFs) in EOC. METHODS: SK-OV-3 and OVCAR-3 cells were used for in vitro study. Following transfection, cell growth and migration were assessed by CCK8 and wound healing assays, respectively. 3'U-tRFs levels were assessed by reverse transcription quantitative PCR (RT-qPCR), following 3'-end RNA polyadenylation. A screening (OVCAD, n = 100) and institutionally independent validation (TU Munich, n = 103) cohorts were employed for survival analysis using disease progression and patients' death as clinical end-points. Bootstrap analysis was performed for internal validation, and decision curve analysis was used to evaluate clinical benefit on disease prognosis. RESULTS: Following primary clinical assessment, target prediction and gene ontology analyses, the 3'U-tRFValCAC (derived from pre-tRNAValCAC) was highlighted to regulate cell proliferation and adhesion, and to correlate with inferior patients' outcome. 3'U-tRFValCAC transfection of SK-OV-3 and OVCAR-3 cells resulted in significantly increased cell growth and migration, in a dose-dependent manner. Elevated tumour 3'U-tRFValCAC levels were associated with significantly higher risk for early progression and worse survival following first-line platinum-based chemotherapy, independently of patients' clinicopathological data, chemotherapy response, and residual tumour. Interestingly, 3'U-tRFValCAC-fitted multivariate models improved risk stratification and provided superior clinical net benefit in prediction of treatment outcome compared to disease established markers. CONCLUSIONS: 3'U-tRFValCAC promotes tumour cell growth and migration and supports modern risk stratification and prognosis in EOC.

Herpes simplex virus alters Alzheimer's disease biomarkers - A hypothesis paper.

INTRODUCTION: Human herpes simplex virus 1 (HSV1) is discussed to induce amyloid-ß (Aß) accumulation and neurofibrillary tangles of hyperphosphorylated tau (pTau) in Alzheimer's disease (AD) in cell culture and animal models. Aß appears to be virostatic. We investigated the association between intrathecal antibodies against HSV or cytomegalovirus (CMV) and cerebrospinal fluid (CSF) AD biomarkers. METHODS: Aß42 /Aß40 ratio, pTau, and tTau were measured in CSF of 117 patients with early AD positive for amyloid pathology (A+) and 30 healthy controls (A-). CSF-to-serum anti-HSV1/2-IgG antibody indices (AI-IgGHSV1/2 ) and CMV (AI-IgGCMV ) were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Exclusively in HSV1-seropositive AD, pTau was positively and significantly predicted by AI-IgGHSV1/2 and negatively by the Aß42 /Aß40 ratio in both univariate and multivariate regression analyses. Furthermore, a significant and negative interaction between the AI-IgGHSV1/2 and Aß42 /Aß40 ratio on pTau was found. DISCUSSION: The results support the hypothesis that HSV infection contributes to AD. HIGHLIGHTS: HSV antibody index is positively associated with tau pathology in patients with AD. HSV antibody index is negatively associated with cerebral FDG metabolism. Amyloid modulates the association of HSV antibody index with CSF-pTau. HSV in AD offers a pathophysiological model connecting tau and amyloid.

Repression of miR-146a in predicting poor treatment outcome in triple-negative breast cancer.

OBJECTIVES: In the era of precision medicine, the highly aggressive and heterogenous triple-negative breast cancer (TNBC) is still characterized by limited options to support personalized prognosis and guide therapeutic interventions. Thereafter, the aim of the present study has been the thorough evaluation of miR-146a as a novel molecular indicator of TNBC prognosis and treatment outcome, utilizing four independent TNBC cohorts. DESIGN & METHODS: miR-146a levels were clinically evaluated in our screening (n = 122) and three external validation TNBC cohorts (de Rinaldis et al. 2013, n = 114; Jézéquel et al. 2015, n = 107; TCGA, n = 180). Analysis of miR-146a and validated gene targets was performed in Jézéquel et al. and TCGA validation cohorts. Patients' survival, recurrence and metastasis were determined as clinical endpoints for the survival analysis. Internal validation was performed by bootstrap analysis and clinical net benefit was evaluated by decision curve analysis. RESULTS: Reduction of miR-146a is strongly associated with patients' poor survival and can predict post-treatment disease early-recurrence, independently of tumor size, lymph node status, histological grade and patients' age. The analysis of the external validation cohorts corroborated the unfavorable nature of miR-146a repression regarding patients' survival and, strikingly, unveiled the ability of miR-146a to predict TNBC metastasis. Combined assessment of miR-146a levels and lymph node status resulted in superior risk-stratification of TNBC patients and higher clinical benefit regarding disease prognosis and post-treatment outcome. Ultimately, miR-146a was negatively associated with EGFR and SOX2 expression in TNBC. CONCLUSIONS: miR-146a evaluation could ameliorate personalized prognosis and support precision medicine decisions in TNBC.

A Pair of Prognostic Biomarkers in Triple-Negative Breast Cancer: KLK10 and KLK11 mRNA Expression.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor patient prognosis and limited therapeutic options. A lack of prognostic biomarkers and therapeutic targets fuels the need for new approaches to tackle this severe disease. Extracellular matrix degradation, release, and modulation of the activity of growth factors/cytokines/chemokines, and the initiation of signaling pathways by extracellular proteolytic networks, have been identified as major processes in the carcinogenesis of breast cancer. Members of the kallikrein-related peptidase (KLK) family contribute to these tumor-relevant processes, and are associated with breast cancer progression and metastasis. In this study, the clinical relevance of mRNA expression of two members of this family, KLK10 and KLK11, has been evaluated in TNBC. For this, their expression levels were quantified in tumor tissue of a large, well-characterized patient cohort (n = 123) via qPCR. Although, in general, the overall expression of both factors are lower in tumor tissue of breast cancer patients (encompassing all subtypes) compared to normal tissue of healthy donors, in the TNBC subtype, expression is even increased. In our cohort, a significant, positive correlation between the expression levels of both KLKs was detected, indicating a coordinate expression mode of these proteases. Elevated KLK10 and KLK11 mRNA levels were associated with poor patient prognosis. Moreover, both factors were found to be independent of other established clinical factors such as age, lymph node status, or residual tumor mass, as determined by multivariable Cox regression analysis. Thus, both proteases, KLK10 and KLK11, may represent unfavorable prognostic factors for TNBC patients and, furthermore, appear as promising potential targets for therapy in TNBC.

Improving the selectivity of 3-amidinophenylalanine-derived matriptase inhibitors.

A rational structure-based approach was employed to develop novel 3-amidinophenylalanine-derived matriptase inhibitors with improved selectivity against thrombin and factor Xa. Of all 23 new derivatives, several monobasic inhibitors exhibit high matriptase affinities and strong selectivity against thrombin. Some inhibitors also possess selectivity against factor Xa, although less pronounced as found for thrombin. A crystal structure of a selective monobasic matriptase inhibitor in complex with matriptase and three crystal structures of related compounds in trypsin and thrombin have been determined. The structures offer an explanation for the different selectivity profiles of these inhibitors and contribute to a more detailed understanding of the observed structure-activity relationship. Selected compounds were tested in vitro against a matriptase-dependent H9N2 influenza virus strain and demonstrated a concentration-dependent inhibition of virus replication in MDCK(II) cells.

CXCL9 inhibits tumour growth and drives anti-PD-L1 therapy in ovarian cancer.

BACKGROUND: Response to immune checkpoint blockade (ICB) in ovarian cancer remains disappointing. Several studies have identified the chemokine CXCL9 as a robust prognosticator of improved survival in ovarian cancer and a characteristic of the immunoreactive subtype, which predicts ICB response. However, the function of CXCL9 in ovarian cancer has been poorly studied. METHODS: Impact of Cxcl9 overexpression in the murine ID8-Trp53-/- and ID8-Trp53-/-Brca2-/- ovarian cancer models on survival, cellular immune composition, PD-L1 expression and anti-PD-L1 therapy. CXCL9 expression analysis in ovarian cancer subtypes and correlation to reported ICB response. RESULTS: CXCL9 overexpression resulted in T-cell accumulation, delayed ascites formation and improved survival, which was dependent on adaptive immune function. In the ICB-resistant mouse model, the chemokine was sufficient to enable a successful anti-PD-L1 therapy. In contrast, these effects were abrogated in Brca2-deficient tumours, most likely due to an already high intrinsic chemokine expression. Finally, in ovarian cancer patients, the clear-cell subtype, known to respond best to ICB, displayed a significantly higher proportion of CXCL9high tumours than the other subtypes. CONCLUSIONS: CXCL9 is a driver of successful ICB in preclinical ovarian cancer. Besides being a feasible predictive biomarker, CXCL9-inducing agents thus represent attractive combination partners to improve ICB in this cancer entity.

Rab31-dependent regulation of transforming growth factor ß expression in breast cancer cells.

BACKGROUND: The small GTP-binding protein Rab31 plays an important role in the modulation of tumor biological-relevant processes, including cell proliferation, adhesion, and invasion. As an underlying mechanism, Rab31 is presumed to act as a molecular switch between a more proliferative and an invasive phenotype. This prompted us to analyze whether Rab31 overexpression in breast cancer cells affects expression of genes involved in epithelial-to-mesenchymal transition (EMT)-like processes when compared to Rab31 low-expressing cells. METHODS: Commercially available profiler PCR arrays were applied to search for differentially expressed genes in Rab31 high- and low-expressing CAMA-1 breast cancer cells. Differential expression of selected candidate genes in response to Rab31 overexpression in CAMA-1 cells was validated by independent qPCR and protein assays. RESULTS: Gene expression profiling of key genes involved in EMT, or its reciprocal process MET, identified 9 genes being significantly up- or down-regulated in Rab31 overexpressing CAMA-1 cells, with the strongest effects seen for TGFB1, encoding TGF-ß1 (> 25-fold down-regulation in Rab31 overexpressing cells). Subsequent validation analyses by qPCR revealed a strong down-regulation of TGFB1 mRNA levels in response to increased Rab31 expression not only in CAMA-1 cells, but also in another breast cancer cell line, MDA-MB-231. Using ELISA and Western blot analysis, a considerable reduction of both intracellular and secreted TGF-ß1 antigen levels was determined in Rab31 overexpressing cells compared to vector control cells. Furthermore, reduced TGF-ß activity was observed upon Rab31 overexpression in CAMA-1 cells using a sensitive TGF-ß bioassay. Finally, the relationship between Rab31 expression and the TGF-ß axis was analyzed by another profiler PCR array focusing on genes involved in TGF-ß signaling. We found 12 out of 84 mRNAs significantly reduced and 7 mRNAs significantly increased upon Rab31 overexpression. CONCLUSIONS: Our results demonstrate that Rab31 is a potent modulator of the expression of TGF-ß and other components of the TGF-ß signaling pathway in breast cancer cells.

PITX2 DNA-Methylation: Predictive versus Prognostic Value for Anthracycline-Based Chemotherapy in Triple-Negative Breast Cancer Patients.

BACKGROUND: PITX2 DNA methylation has been shown to predict outcomes in high-risk breast cancer patients after anthracycline-based chemotherapy. To determine its prognostic versus predictive value, the impact of PITX2 DNA methylation on outcomes was studied in an untreated cohort vs. an anthracycline-treated triple-negative breast cancer (TNBC) cohort. MATERIAL AND METHODS: The percent DNA methylation ratio (PMR) of paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time PCR test. Patient samples of routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue and clinical data from 144 TNBC patients of 2 independent cohorts (i.e., 66 untreated patients and 78 patients treated with anthracycline-based chemotherapy) were analyzed. RESULTS: The risk of 5- and 10-year overall survival (OS) increased continuously with rising PITX2 DNA methylation in the anthracycline-treated population, but it increased only slightly during 10-year follow-up time in the untreated patient population. PITX2 DNA methylation with a PMR cutoff of 2 did not show significance for poor vs. good outcomes (OS) in the untreated patient cohort (HR = 1.55; p = 0.259). In contrast, the PITX2 PMR cutoff of 2 identified patients with poor (PMR >2) vs. good (PMR ≤2) outcomes (OS) with statistical significance in the anthracycline-treated cohort (HR = 3.96; p = 0.011). The results in the subgroup of patients who did receive anthracyclines only (no taxanes) confirmed this finding (HR = 5.71; p = 0.014). CONCLUSION: In this hypothesis-generating study PITX2 DNA methylation demonstrated predominantly predictive value in anthracycline treatment in TNBC patients. The risk of poor outcome (OS) correlates with increasing PITX2 DNA methylation.

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma.

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.

The prognostic and diagnostic potential of kallikrein-related peptidases in ovarian cancer.

Introduction: Ovarian cancer is one of the deadliest malignancies among women worldwide. The lack of early diagnostic markers fuels an unfavorable prognosis as most patients are at an advanced stage when the disease is diagnosed for the first time. The role of the kallikrein-related peptidase (KLK) family in ovarian cancer progression and prognosis has been thoroughly investigated in various studies. Most of these peptidases are upregulated in ovarian cancer tissue compared to normal ovarian tissue and their expression is linked to overall and progression-free survival (OS/PFS). In this review, we address the clinical relevance of KLKs in ovarian cancer and their diagnostic potential.Areas covered: This review covers the expression and regulation of KLKs in ovarian cancer with focus on the prognostic and diagnostic potential, especially in liquid biopsies.Expert opinion: In ovarian cancer, several kallikrein-related peptidases are markedly expressed in a tissue-specific manner. Tumor-derived protease secretion results in elevated levels in serum and ascites. KLKs may thus serve as potential biomarkers alone or in combination with other serum tumor markers, such as Cancer Antigen 125 (CA125), for early detection and assessment of the prognosis of ovarian cancer patients.

Elevated levels of both microRNA 378 (miR-378) and kallikrein-related peptidase 4 (KLK4) mRNA are associated with an unfavorable prognosis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) patients have the worst outcome among all breast cancer subtypes. In oral squamous carcinoma cells, miR-378 was reported to target the mRNA of kallikrein-related peptidase 4 (KLK4), resulting in inhibition of cell proliferation, migration and invasion, induction of apoptosis, and reduction of tumor growth in vivo. Similarly, a miR-378/KLK4 axis has been proposed in prostate cancer. Here, we analyzed the correlation between miR-378 and KLK4 mRNA expression and determined the prognostic impact of both factors in TNBC. miR-378 and KLK4 mRNA expression levels were determined by quantitative PCR in tumor tissue of TNBC patients (n=103) and correlated with clinical parameters and patients' survival. There was no significant correlation between miR-378 and KLK4 mRNA expression. In univariate Cox regression analysis, elevated miR-378 expression was significantly associated with shortened disease-free survival (DFS, P=0.047) and overall survival (OS, P=0.031), high KLK4 mRNA levels were linked to a worse DFS (P=0.033). Combination of KLK4 mRNA and miR-378 (KLK4+miR-378, low/low versus high and/or high) allowed even better discrimination between favorable and unfavorable prognosis (DFS, P=0.008; OS, P=0.025). In multivariable analysis, miR-378 and KLK4+miR-378 expression remained independent predictive factors for DFS (P=0.014, P=0.010, respectively) and OS (P=0.016, P=0.049, respectively), while KLK4 mRNA only showed a trend towards significance for DFS (P=0.061). Our findings suggest that in TNBC there is no significant impact of miR-378 on KLK4 expression. Both factors, miR-378 and, to a lesser extent, KLK4 mRNA represent unfavorable prognostic markers in TNBC patients.

Substrate-biased activity-based probes identify proteases that cleave receptor CDCP1.

CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

The Chemokine CX3CL1 Improves Trastuzumab Efficacy in HER2 Low-Expressing Cancer In Vitro and In Vivo.

A crucial mode of action of trastuzumab is the labeling of HER2-positive (HER2+) tumor cells for the eradication by natural killer (NK) cells, a process called antibody-dependent cellular cytotoxicity (ADCC). However, despite widespread HER2 expression among cancer entities, only a fraction, with robust HER2 overexpression, benefits from trastuzumab therapy. ADCC requires both sufficient lymphocytic infiltration and close binding of the immune cells to the antibody-tagged tumor cells. We hypothesized that the chemokine CX3CL1 could improve both processes, as it is synthesized as a membrane-bound, adhesive form that is eventually cleaved into a soluble, chemotactic protein. Here, we show that CX3CL1 overexpression is a positive prognostic marker in breast cancer. CX3CL1 overexpression attracted tumor-suppressive lymphocytes, including NK cells, and inhibited tumor growth and lung metastasis in the syngeneic 4T1 breast cancer mouse model. In HER2+ SKBR3, MDA-MB-453, and HT-29 tumor cells, CX3CL1 overexpression increased NK cell-mediated cytotoxicity in vitro and acted synergistically with trastuzumab. Even though CX3CL1 did not further improve trastuzumab efficacy in vivo in the trastuzumab-sensitive MDA-MB-453 model, it compensated for NK-cell depletion and prolonged survival. In the HER2 low-expressing HT-29 model, however, CX3CL1 overexpression not only prolonged survival time but also overcame trastuzumab resistance in a partly NK cell-dependent manner. Taken together, these findings identify CX3CL1 as a feasible pharmacologic target to enable trastuzumab therapy in HER2 low-expressing cancers and render it a potential predictive biomarker to determine therapy responders.

Epigenetic modulators combination with chemotherapy in breast cancer cells.

Despite the concerning adverse effects on tumour development, epigenetic drugs are very promising in cancer treatment. The aim of this study was to compare the differential effects of standard chemotherapy regimens (FEC: 5-fluorouracil plus epirubicine plus cyclophosphamide) in combination with epigenetic modulators (decitabine, valproic acid): (a) on gene methylation levels of selected tumour biomarkers (LINE-1, uPA, PAI-1, DAPK); (b) their expression status (uPA and PAI-1); (c) differentiation status (5meC and H3K27me3). Furthermore, cell survival as well as changes concerning the invasion capacity were monitored in cell culture models of breast cancer (MCF-7, MDA-MB-231). A significant overall decrease of cell survival was observed in the FEC-containing combination therapies for both cell lines. Methylation results showed a general tendency towards increased demethylation of the uPA and PAI-1 gene promoters for the MCF-7 cells, as well as the proapoptotic DAPK gene in the treatment regimens for both cell lines. The uPA and PAI-1 antigen levels were mainly increased in the supernatant of FEC-only treated MDA-MB-231 cells. DAC-only treatment induced an increase of secreted uPA protein in MCF-7 cell culture, while most of the VPA-containing regimens also induced uPA and PAI-1 expression in MCF-7 cell fractions. Epigenetically active substances can also induce a re-differentiation in tumour cells, as shown by 5meC, H3K27me3 applying ICC. SIGNIFICANCE OF THE STUDY: Epigenetic modulators especially in the highly undifferentiated and highly malignant MDA-MB-231 tumour cells significantly reduced tumour malignancy thus; further clinical studies applying specific combination therapies with epigenetic modulators may be warranted.

tRNAGlyGCC-Derived Internal Fragment (i-tRF-GlyGCC) in Ovarian Cancer Treatment Outcome and Progression.

Epithelial ovarian cancer (EOC) remains a highly-lethal gynecological malignancy, characterized by frequent recurrence, chemotherapy resistance and poor 5-year survival. Identifying novel predictive molecular markers remains an overdue challenge in the disease's clinical management. Herein, in silico analysis of TCGA-OV highlighted the tRNA-derived internal fragment (i-tRF-GlyGCC) among the most abundant tRFs in ovarian tumors, while target prediction and gene ontology (GO) enrichment analysis predicted its implication in key biological processes. Thereafter, i-tRF-GlyGCC levels were quantified in a screening EOC (n = 98) and an institutionally-independent serous ovarian cancer (SOC) validation cohort (n = 100, OVCAD multicenter study). Disease progression and patient death were used as clinical endpoints for the survival analysis. Internal validation was performed by bootstrap analysis and the clinical net benefit was estimated by decision curve analysis. The analysis highlighted the significant association of i-tRF-GlyGCC with advanced FIGO stages, suboptimal debulking and most importantly, with early progression and poor overall survival of EOC patients. The OVCAD validation cohort corroborated the unfavorable predictive value of i-tRF-GlyGCC in EOC. Ultimately, evaluation of i-tRF-GlyGCC with the established/clinically used prognostic markers offered superior patient risk-stratification and enhanced clinical benefit in EOC prognosis. In conclusion, i-tRF-GlyGCC assessment could aid towards personalized prognosis and support precision medicine decisions in EOC.

KLK4 Induces Anti-Tumor Effects in Human Xenograft Mouse Models of Orthotopic and Metastatic Prostate Cancer.

Recent reports have suggested the role of kallikrein-related peptidase 4 (KLK4) to be that of remodeling the tumor microenvironment in many cancers, including prostate cancer. Notably, these studies have suggested a pro-tumorigenic role for KLK4, especially in prostate cancer. However, these have been primarily in vitro studies, with limited in vivo studies performed to date. Herein, we employed an orthotopic inoculation xenograft model to mimic the growth of primary tumors, and an intracardiac injection to induce metastatic dissemination to determine the in vivo tumorigenic effects of KLK4 overexpressed in PC3 prostate cancer cells. Notably, we found that these KLK4-expressing cells gave rise to smaller localized tumors and decreased metastases than the parent PC-3 cells. To our knowledge, this is the first report of an anti-tumorigenic effect of KLK4, particularly in prostate cancer. These findings also provide a cautionary tale of the need for in vivo analyses to substantiate in vitro experimental data.

Prognostic value of kallikrein-related peptidase 7 (KLK7) mRNA expression in advanced high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer. A growing body of evidence suggests tumor-supporting roles of several members of the kallikrein-related peptidase (KLK) family, including KLK5 and KLK7, in this cancer subtype. In normal physiology, KLK5 and KLK7 are the major proteases involved in skin desquamation. Moreover, in several cancer types KLK5 and KLK7 co-expression has been observed. Recently, we have shown that elevated KLK5 mRNA levels are associated with an unfavorable prognosis in HGSOC. Therefore, the aim of this study was to investigate the clinical significance of KLK7 mRNA expression and to explore its relation to KLK5 levels in HGSOC. METHODS: mRNA expression levels of KLK7 were quantified by qPCR in a well-characterized patient cohort afflicted with advanced high-grade serous ovarian cancer (FIGO III/IV, n = 139). Previously determined KLK5 mRNA as well as KLK5 and KLK7 antigen concentrations were used to evaluate the relationship between the expression patterns of both factors on the mRNA as well as protein level in tumor tissue of HGSOC patients. RESULTS: There were strong, significant positive correlations between KLK5 and KLK7 both at the mRNA and the protein level, suggesting coordinate expression of these proteases in HGSOC. In univariate analyses, elevated KLK7 levels as well as the combination of KLK5 + KLK7 (high and/or high versus low/low) were significantly associated with worse progression-free survival (PFS). High mRNA expression levels of KLK7 and the combination of KLK5 and KLK7 showed a trend towards significance for overall survival (OS). In multivariate analyses, KLK7 mRNA expression represented an unfavorable, statistically significant independent predictor for PFS and OS. CONCLUSIONS: The findings imply that both increased KLK5 and KLK7 mRNA expression levels represent unfavorable prognostic biomarkers in advanced high-grade serous ovarian cancer, whereby multivariate analyses indicate that KLK7 mRNA exhibits a stronger predictive value as compared to KLK5 mRNA and the combination of KLK5 and KLK7.

High levels of KLK7 protein expression are related to a favorable prognosis in triple-negative breast cancer patients.

In normal physiology, kallikrein-related peptidase 7 (KLK7), together with other members of the kallikrein-related peptidase family, is mainly involved in skin desquamation and keratinization processes. Moreover, expression of KLK7 was shown in various tumor types to be dysregulated and to correlate to patients' survival time. However, there are contradictory reports in breast cancer whether KLK7 represents an unfavorable or favorable prognostic biomarker. In the present study, we examined the prognostic value of KLK7 protein expression in triple-negative breast cancer (TNBC), determined by immunohistochemistry (IHC). A cohort encompassing 133 TNBC specimens, present on tissue microarrays, was analyzed. For quantification of the staining intensity, an automated digital IHC image analysis algorithm was applied. In both Kaplan-Meier and univariate Cox analyses, elevated KLK7 protein levels were significantly linked with prolonged overall survival (OS). In multivariable Cox analysis, addition of KLK7 immunoreactivity scores to the base model (including the clinical parameters age, tumor size, and nodal status) demonstrated that KLK7 protein expression remained as a statistically significant, independent parameter for prolonged OS. These results strongly indicate that KLK7 is a favorable prognostic biomarker in triple-negative breast cancer.

miR-181a overexpression predicts the poor treatment response and early-progression of serous ovarian cancer patients.

Ovarian cancer (OC) remains a leading cause of gynecological cancer-related death worldwide, characterized by poor 5-year survival. Molecular markers could serve as crucial tools of personalized prognosis and therapy. Herein, we present miR-181a as novel predictor of OC prognosis, using five independent OC cohorts. In particular, a screening (n = 81) and an institutionally independent validation (n = 100, OVCAD multicenter study) serous OC (SOC) cohorts were analyzed. Bagnoli et al (2016) OC179 (n = 124) to OC133 (n = 100) and TCGA (n = 489) served as external validation cohorts. Patients' survival and disease progression were assessed as clinical endpoint events. Bootstrap analysis was performed for internal validation and decision curve analysis was utilized to evaluate clinical benefit. miR-181a overexpression was unveiled as powerful and independent molecular predictor of patients' poor survival and higher risk for disease progression after debulking surgery and platinum-based chemotherapy. Analysis of the OVCAD institutionally independent cohort, as well as of Bagnoli et al. and TCGA external cohorts further confirmed the unfavorable prognostic nature of miR-181a overexpression in SOC. Strikingly, multivariate prognostic models incorporating miR-181a with established disease markers clearly improved patients' risk-stratification and offered superior clinical benefit in OC prognostication. Conclusively, miR-181a evaluation could augment prognostic accuracy and support precision medicine decisions in OC.