miRNA dynamics in tumor-infiltrating myeloid cells modulating tumor progression in pancreatic cancer.

Myeloid cells including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are known as important mediators of tumor progression in solid tumors such as pancreatic cancer. Infiltrating myeloid cells have been identified not only in invasive tumors, but also in early pre-invasive pancreatic intraepithelial precursor lesions (PanIN). The functional dynamics of myeloid cells during carcinogenesis is largely unknown. We aimed to systematically elucidate phenotypic and transcriptional changes in infiltrating myeloid cells during carcinogenesis and tumor progression in a genetic mouse model of pancreatic cancer. Using murine pancreatic myeloid cells isolated from the genetic mouse model at different time points during carcinogenesis, we examined both established markers of macrophage polarization using RT-PCR and FACS as well as transcriptional changes focusing on miRNA profiling. Myeloid cells isolated during carcinogenesis showed a simultaneous increase of established markers of M1 and M2 polarization during carcinogenesis, indicating that phenotypic changes of myeloid cells during carcinogenesis do not follow the established M1/M2 classification. MiRNA profiling revealed distinct regulations of several miRNAs already present in myeloid cells infiltrating pre-invasive PanIN lesions. Among them miRNA-21 was significantly increased in myeloid cells surrounding both PanIN lesions and invasive cancers. Functionally, miRNA-21-5p and -3p altered expression of the immune-modulating cytokines CXCL-10 and CCL-3 respectively. Our data indicate that miRNAs are dynamically regulated in infiltrating myeloid cells during carcinogenesis and mediate their functional phenotype by facilitating an immune-suppressive tumor-promoting micro-milieu.

CUX1: a modulator of tumour aggressiveness in pancreatic neuroendocrine neoplasms.

Pancreatic neuroendocrine neoplasms (PNENs) constitute a rare tumour entity, and prognosis and treatment options depend on tumour-mediating hallmarks such as angiogenesis, proliferation rate and resistance to apoptosis. The molecular pathways that determine the malignant phenotype are still insufficiently understood and this has limited the use of effective combination therapies in the past. In this study, we aimed to characterise the effect of the oncogenic transcription factor Cut homeobox 1 (CUX1) on proliferation, resistance to apoptosis and angiogenesis in murine and human PNENs. The expression and function of CUX1 were analysed using knockdown and overexpression strategies in Ins-1 and Bon-1 cells, xenograft models and a genetically engineered mouse model of insulinoma (RIP1Tag2). Regulation of angiogenesis was assessed using RNA profiling and functional tube-formation assays in HMEC-1 cells. Finally, CUX1 expression was assessed in a tissue microarray of 59 human insulinomas and correlated with clinicopathological data. CUX1 expression was upregulated during tumour progression in a time- and stage-dependent manner in the RIP1Tag2 model, and associated with pro-invasive and metastatic features of human insulinomas. Endogenous and recombinant CUX1 expression increased tumour cell proliferation, tumour growth, resistance to apoptosis, and angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of CUX1 was mediated via upregulation of effectors such as HIF1α and MMP9. CUX1 mediates an invasive pro-angiogenic phenotype and is associated with malignant behaviour in human insulinomas.

Synthetic lethality screen identifies RPS6KA2 as modifier of epidermal growth factor receptor activity in pancreatic cancer.

Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR) inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA)-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2)/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.

Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells.

Hydrophobins are fungal proteins with the ability to form immunologically inert membranes of high stability, properties that makes them attractive candidates for orthopaedic implant coatings. Cell adhesion on the surface of such implants is necessary for better integration with the neighbouring tissue; however, hydrophobin surfaces do not mediate cell adhesion. The aim of this project was therefore to investigate whether the class I hydrophobin DewA from Aspergillus nidulans can be functionalized for use on orthopaedic implant surfaces. DewA variants bearing either one RGD sequence or the laminin globular domain LG3 binding motif were engineered. The surfaces of both variants showed significantly increased adhesion of mesenchymal stem cells (MSCs), osteoblasts, fibroblasts and chondrocytes; in contrast, the insertion of binding motifs RGD and LG3 in DewA did not increase Staphylococcus aureus adhesion to the hydrophobin surfaces. Proliferation of MSCs and their osteogenic, chondrogenic and adipogenic differentiation potential were not affected on these surfaces. The engineered surfaces therefore enhanced MSC adhesion without interfering with their functionality or leading to increased risk of bacterial infection.