Mervi Toriseva

Matrix metalloproteinase-19 is expressed by proliferating epithelium but disappears with neoplastic dedifferentiation.

MMP‐19 (also designated RASI) is a recently discovered member of a large family of zinc‐dependent proteolytic enzymes, most of which have been implicated in cancer growth and metastasis. It differs from the others by its chromosomal location and structure and is expressed by endothelial and vascular smooth muscle cells in vivo. Our aim was to study the putative role of MMP‐19 in skin cancer. We also examined its regulation in keratinocyte cultures using quantitative TaqMan RT‐PCR. Our results show that MMP‐19 can also be detected in stimulated keratinocytes by Northern and Western analyses. In wounds, it was found in keratinocytes outside the migrating area, while in BCC and SCC, it was present in the hyperproliferative (p63‐positive), E‐cadherin‐negative epidermis at the tumor surface but downregulated in invasive cancer islands. Expression was also evident in endothelial cells of neoangiogenic regions and in occasional stromal fibroblasts. Of the 12 tested cytokines/growth factors, only TNF‐α and PMA were able to stimulate the expression of MMP‐19 mRNA in primary keratinocytes. No MMP‐19 mRNA was detected by Northern analysis in cultured HaCaT or A5 cells or in an SCC cell line established from head‐and‐neck cancer. Our data suggest that, unlike most MMPs, MMP‐19 expression in the epidermis is downregulated during transformation and histologic dedifferentiation.

MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13 −/−) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13 −/− mice. Granulation tissue in Mmp13 −/− mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13 −/− mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13−/− mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13 −/− granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13 −/− mice compared to WT mice. Mmp13 −/− mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.

Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention

Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro.

p38δ mitogen-activated protein kinase regulates the expression of tight junction protein ZO-1 in differentiating human epidermal keratinocytes

Increasing evidence has recognized tight junctions (TJs) as the lower epidermal inside-out diffusion barrier located in granular cell layers of the epidermis. However, little is known about the regulation of TJ components in epidermis. p38 pathway is one of the mitogen-activated protein kinase pathways, which controls cell growth, differentiation, and apoptosis. We have investigated the role of p38 signaling pathway in the regulation of selected desmosomal, adherens and TJ components in human primary keratinocytes during Ca2+-induced differentiation, as well as in cultured squamous cell carcinoma cell lines. p38 signaling pathway was inhibited in cultured keratinocytes and cutaneous squamous cell carcinoma cells using recombinant adenoviruses, small inhibitory RNAs (siRNA) and chemical inhibitors. Expression of intercellular junction proteins was investigated using Western analysis and indirect immunofluorescence (IIF). The results showed that inhibition of p38δ function by siRNA or adenovirally delivered dominant negative mutant led to markedly decreased levels of Zonula occludens-1 (ZO-1) protein in keratinocytes, while the expression of other junctional proteins studied was not altered. Immunolocalization of ZO-1 revealed that intercellular junction areas were depleted from ZO-1. Inhibition of ZO-1 by siRNA silencing did not however result in an altered expression or subcellular localization of other TJ components studied. The expression of ZO-1 in carcinoma cells was also regulated by p38. The results indicate that ZO-1 is regulated by p38δ while the other junction proteins studied are not. Since ZO-1 is an integral component of functional TJs, various pathological processes affecting signaling via p38δ may also interfere with epithelial maturation and the formation and function of TJs.

Keratinocyte growth factor induces gene expression signature associated with suppression of malignant phenotype of cutaneous squamous carcinoma cells.

Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. We have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n = 6) than in normal skin samples (n = 6). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but rather suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal keratinocytes.

EphB2 Promotes Progression of Cutaneous Squamous Cell Carcinoma.

Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo. Tumor cell-specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability, migration of tumor cells, and invasion of tumor cells. Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13. Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer.

Increased expression of fibroblast growth factor 13 in prostate cancer is associated with shortened time to biochemical recurrence after radical prostatectomy

Fibroblast growth factor homologous factors (FHFs) belong to the fibroblast growth factor (FGF) superfamily, which plays an important role in prostate cancer (PCa). Mining of public database suggests that FGF13 (FHF2) mRNA expression is altered in over 30% of PCa cases. This study examined the FGF13 expression pattern in human PCa specimens and evaluated its potential as a biomarker for patient outcome after radical prostatectomy (RP). Immunohistochemistry (IHC) showed that FGF13 was detectable in the majority of human PCa samples, and FGF13 IHC scores were higher in high‐grade prostatic intraepithelial neoplasia, in primary PCa and in metastatic PCa than in benign prostatic tissue. There was a significant association between high cytoplasmic FGF13 staining and a risk of biochemical recurrence (BCR) after RP. This was also evident in the intermediate to high‐risk patient groups. In contrast, positive nuclear FGF13 staining along with low cytoplasmic FGF13 group showed a decreased BCR risk. Multivariate regression analysis indicated that high cytoplasmic FGF13 staining was associated with BCR and that this could serve as an independent prognostic marker in PCa. Several PCa cell lines showed increased FGF13 expression at the mRNA and protein levels compared to the immortalized prostate epithelial cell line PNT1a. Analysis of co‐labeled cells suggested a possible interaction of FGF13 with α‐tubulin and the voltage‐gated sodium channel proteins (NaVs/VGSCs). Our data indicate that, for PCa patients after RP, FGF13 serves as a potential novel prognostic marker that improves prediction of BCR‐free survival, in particular if combined with other clinical parameters.

A high-content image analysis approach for quantitative measurements of chemosensitivity in patient-derived tumor microtissues

Organotypic, three-dimensional (3D) cancer models have enabled investigations of complex microtissues in increasingly realistic conditions. However, a drawback of these advanced models remains the poor biological relevance of cancer cell lines, while higher clinical significance would be obtainable with patient-derived cell cultures. Here, we describe the generation and data analysis of 3D microtissue models from patient-derived xenografts (PDX) of non-small cell lung carcinoma (NSCLC). Standard of care anti-cancer drugs were applied and the altered multicellular morphologies were captured by confocal microscopy, followed by automated image analyses to quantitatively measure phenotypic features for high-content chemosensitivity tests. The obtained image data were thresholded using a local entropy filter after which the image foreground was split into local regions, for a supervised classification into tumor or fibroblast cell types. Robust statistical methods were applied to evaluate treatment effects on growth and morphology. Both novel and existing computational approaches were compared at each step, while prioritizing high experimental throughput. Docetaxel was found to be the most effective drug that blocked both tumor growth and invasion. These effects were also validated in PDX tumors in vivo. Our research opens new avenues for high-content drug screening based on patient-derived cell cultures, and for personalized chemosensitivity testing.

Quantitative Phenotypic Image Analysis of Three-Dimensional Organotypic Cultures

Glandular epithelial cells differentiate into three-dimensional (3D) multicellular or acinar structures, particularly when embedded in laminin-rich extracellular matrix (ECM). The spectrum of different multicellular morphologies formed in 3D is a reliable indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. Motile cancer cells may actively invade the matrix, utilizing epithelial, mesenchymal, or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are also very sensitive to small-molecule inhibitors that, e.g., target the actin cytoskeleton. Our strategy is to recapitulate the formation and the histology of complex solid cancer tissues in vitro, based on cell culture technologies that promote the intrinsic differentiation potential of normal and transformed epithelial cells, and also including stromal fibroblasts and other key components of the tumor microenvironment. We have developed a streamlined stand-alone software solution that supports the detailed quantitative phenotypic analysis of organotypic 3D cultures. This approach utilizes the power of automated image analysis as a phenotypic readout in cell-based assays. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of a large number of multicellular structures, which can form a multitude of different organoid shapes, sizes, and textures according to their capacity to engage in epithelial differentiation programs or not. At the far end of this spectrum of tumor-relevant differentiation properties, there are highly invasive tumor cells or multicellular structures that may rapidly invade the surrounding ECM, but fail to form higher-order epithelial tissue structures. Furthermore, this system allows us to monitor dynamic changes that can result from the extraordinary plasticity of tumor cells, e.g., epithelial-to-mesenchymal transition in live cell settings. Furthermore, AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate cell-based organotypic 3D assays in basic research, drug discovery, and target validation.

Abstract 4951: FGFRL1 in prostate cancer progression

Prostate cancer (PCa) is a disease with high incidence, however, many PCa patients are over-diagnosed and over-treated. Molecular characterization of PCa provides a valid approach to stratify patients, and thus reduce overtreatment. Fibroblast growth factors and their receptor (FGF/FGFR) signaling pathways are involved in various cellular functions such as proliferation, differentiation, migration, and apoptosis of prostate cancer cells. Dysregulated and constitutively activated FGF/FGFR pathways have been shown to be involved in the initiation and progression of prostate cancer. Fibroblast growth factor receptor like 1 (FGFRL1, FGFR5) is the most recently identified member of the FGFR family. FGFRL1 binds several FGFs but its short intracellular part lacks a tyrosine kinase domain. Therefore, the extracellular domain has been suggested to act as a dominant negative regulator of other FGFRs. However, cellular functions of FGFRL1 remain poorly understood. Aberrant FGFRL1 expression has been reported in ovarian, bladder, colon, and other cancers. In silico data analysis indicated altered FGFRL1 mRNA expression in 17% of PCa cases. To date, there have been no systematic studies of FGFRL1 expression and function in prostate and PCa. We studied FGFRL1 expression and function in PCa cell lines, xenografts and in human PCa specimens. FGRL1 was knocked-down in PC-3M cells, by shRNAs, which showed reduced growth as nude mouse xenografts when compared to control. This argues against a dominant negative function. To study FGFRL1 in human PCas, we collected formalin-fixed paraffin-embedded samples from PCa patients undergoing radical prostatectomy (n = 243), metastatic PCa (n = 36) and castration-resistant PCa (n = 21). Samples were prepared for mRNA analysis, and immunohistochemistry. The overall levels of FGFRL1 immunostaining were significantly increased in PCa compared to normal tissue. In normal tissue, FGFRL1 immunostaining localized to the cell membrane and to a lesser extent to the cytoplasm and nuclei. In PCa, protein expression of FGFRL1 was decreased at the cell membrane, while expression in the cytoplasm and nucleus were increased. Low membrane and high nuclear immunostaining of FGFRL1 correlated with high Gleason grade. High nuclear FGFRL1 also correlated with high levels of preoperative serum PSA and an increased proportion of tumors with positive surgical margins. Our results suggest that FGFRL1 may play an active role in PCa cells and in tumor progression and can possibly be used to assess PCa prognosis. Citation Format: Lan Yu, Andrew Erickson, Mervi Toriseva, Teresa Elo, Johanna Tuomela, Heikki Seikkula, Martti Nurmi, Peter Bostrom, Tuomas Mirtti, Kalle Alanen, Markku Kallajoki, Matthias Nees, Pirkko Harkonen. FGFRL1 in prostate cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4951.