Tribbles Genes in Gastric Cancer: A Tumor-Suppressive Role for TRIB2.

Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients' outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype.

Combined Targeting of Pathogenetic Mechanisms in Pancreatic Neuroendocrine Tumors Elicits Synergistic Antitumor Effects.

Pancreatic neuroendocrine neoplasms (PanNENs) are the second most common malignancy of the pancreas. Surgery remains the only curative treatment for localized disease. For patients with inoperable advanced or metastatic disease, few targeted therapies are available, but their efficacy is unpredictable and variable. Exploiting prior knowledge on pathogenetic processes involved in PanNEN tumorigenesis, we tested buparlisib (PI3K inhibitor) and ribociclib (CDK4/6 inhibitor), as single agents or in combination, in different preclinical models. First, we used cell lines representative of well-differentiated (INS-1E, NT-3) and poorly differentiated (BON-1) PanNENs. The combination of buparlisib with ribociclib reduced the proliferation of 2D and 3D spheroid cultures more potently than the individual drugs. Buparlisib, but not ribociclib, induced apoptosis. The anti-proliferative activity of the drugs correlated with downstream target inhibition at mRNA and protein levels. We then tested the drugs on primary islet microtissues from a genetic PanNET animal model (Men1-defective mice) and from wild-type mice: the drug combination was effective against the former without altering islet cell physiology. Finally, we treated PanNET patient-derived islet-like 3D tumoroids: the combination of buparlisib with ribociclib was effective in three out of four samples. Combined targeting of PI3K and CDK4/6 is a promising strategy for PanNENs spanning various molecular and histo-pathological features.

HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer.

In this study, two novel alternative splice variants of HER2, named HER2-PI9 and HER2-I12, were identified in breast cancer cell lines and breast tumour tissues. Whilst HER2-P19 arises from the inclusion of an 117 bp cassette-exon of intron 9 of HER2, HER2-I12 results from intron 12 inclusion. In silico analyses were performed to predict the amino acid sequences of these two HER2 novel variants. To confirm their protein expression, plasmid vectors were generated and transfected into the HER2 negative breast cancer cell line, MCF-7. Additionally, their functional properties in oncogenic signalling were confirmed. Expression of HER2-PI9 and HER2-I12 was successful and matched the in silico predictions. Importantly, these splice variants can modulate the phosphorylation levels of extracellular signal-related kinase 1/2 (ERK1/2) and Akt/protein kinase B (Akt) signalling in MCF-7 breast cancer cells. Enhanced cellular proliferation, migration and invasion were observed in the case of the HER2-I12 expressing model. In human tissues and breast carcinoma tumours both variants were present. This study reveals two novel splice variants of HER2. Additionally, the potential biological activity for HER2-PI9 and HER2-I12 in breast cancer cells is also reported..

Tribbles-1 Expression and Its Function to Control Inflammatory Cytokines, Including Interleukin-8 Levels are Regulated by miRNAs in Macrophages and Prostate Cancer Cells.

The pseudokinase TRIB1 controls cell function in a range of contexts, by regulating MAP kinase activation and mediating protein degradation via the COP1 ubiquitin ligase. TRIB1 regulates polarization of macrophages and dysregulated Trib1 expression in murine models has been shown to alter atherosclerosis burden and adipose homeostasis. Recently, TRIB1 has also been implicated in the pathogenesis of prostate cancer, where it is often overexpressed, even in the absence of genetic amplification. Well described TRIB1 effectors include MAP kinases and C/EBP transcription factors, both in immune cells and in carcinogenesis. However, the mechanisms that regulate TRIB1 itself remain elusive. Here, we show that the long and conserved 3'untranslated region (3'UTR) of TRIB1 is targeted by miRNAs in macrophage and prostate cancer models. By using a systematic in silico analysis, we identified multiple "high confidence" miRNAs potentially binding to the 3'UTR of TRIB1 and report that miR-101-3p and miR-132-3p are direct regulators of TRIB1 expression and function. Binding of miR-101-3p and miR-132-3p to the 3'UTR of TRIB1 mRNA leads to an increased transcription and secretion of interleukin-8. Our data demonstrate that modulation of TRIB1 by miRNAs alters the inflammatory profile of both human macrophages and prostate cancer cells.

How is Herstatin, a tumor suppressor splice variant of the oncogene HER2, regulated?

The human epidermal growth factor receptor 2 (HER2)/receptor tyrosine-protein kinasebB-2 (ERBB2) is overexpressed in 20-30% of breast tumors leading to faster growing and more aggressive tumors. Alternative splicing generates a functionally distinct HER2 variant called Herstatin, which is produced by the inclusion of intron 8. Herstatin acts as a tumor suppressor by effectively blocking HER2 activity and cell proliferation, while promoting apoptosis. In the present study we investigated HER2 pre-mRNA regulatory sequences and splicing factors which regulate the alternative splicing of Herstatin. A Herstatin minigene, comprising exon 8/intron 8/exon 9 of HER2 was generated and subsequent in vitro splicing assays revealed that RNA secondary structure and somatic mutations did not impact on inclusion of intron 8. However, using RNase-assisted RNA chromatography, followed by mass spectrometry, we identified six RNA-binding proteins (splicing factors) that bind to RNA sequences surrounding exon 8/intron 8 and intron 8/exon 9 boundaries; these included hnRNP I, H1, D, A2/B1 and hnRNPA1 plus the SR protein SRSF1. Specifically, overexpression of hnRNP A1 significantly increased retention of intron 8 resulting in higher levels of Herstatin in SKBR3 breast cancer cells whereas SRSF1 only had a marginal effect in decreasing Herstatin but increased exogenous HER2 levels under these experimental conditions. In conclusion, we have identified the first splicing factors and regulatory sequences that are involved in the production of Herstatin.