Annotation and functional characterization of long noncoding RNAs deregulated in pancreatic adenocarcinoma.

PURPOSE: Transcriptome analysis of pancreatic ductal adenocarcinoma (PDAC) has been useful to identify gene expression changes that sustain malignant phenotypes. Yet, most studies examined only tumor tissues and focused on protein-coding genes, leaving long non-coding RNAs (lncRNAs) largely underexplored. METHODS: We generated total RNA-Seq data from patient-matched tumor and nonmalignant pancreatic tissues and implemented a computational pipeline to survey known and novel lncRNAs. siRNA-mediated knockdown in tumor cell lines was performed to assess the contribution of PDAC-associated lncRNAs to malignant phenotypes. Gene co-expression network and functional enrichment analyses were used to assign deregulated lncRNAs to biological processes and molecular pathways. RESULTS: We detected 9,032 GENCODE lncRNAs as well as 523 unannotated lncRNAs, including transcripts significantly associated with patient outcome. Aberrant expression of a subset of novel and known lncRNAs was confirmed in patient samples and cell lines. siRNA-mediated knockdown of a subset of these lncRNAs (LINC01559, LINC01133, CCAT1, LINC00920 and UCA1) reduced cell proliferation, migration and invasion. Gene co-expression network analysis associated PDAC-deregulated lncRNAs with diverse biological processes, such as cell adhesion, protein glycosylation and DNA repair. Furthermore, UCA1 knockdown was shown to specifically deregulate co-expressed genes involved in DNA repair and to negatively impact DNA repair following damage induced by ionizing radiation. CONCLUSIONS: Our study expands the repertoire of lncRNAs deregulated in PDAC, thereby revealing novel candidate biomarkers for patient risk stratification. It also provides a roadmap for functional assays aimed to characterize novel mechanisms of action of lncRNAs in pancreatic cancer, which could be explored for therapeutic development.

IKKß Kinase Promotes Stemness, Migration, and Invasion in KRAS-Driven Lung Adenocarcinoma Cells.

KRAS oncogenic mutations are widespread in lung cancer and, because direct targeting of KRAS has proven to be challenging, KRAS-driven cancers lack effective therapies. One alternative strategy for developing KRAS targeted therapies is to identify downstream targets involved in promoting important malignant features, such as the acquisition of a cancer stem-like and metastatic phenotype. Based on previous studies showing that KRAS activates nuclear factor kappa-B (NF-κB) through inhibitor of nuclear factor kappa-B kinase ß (IKKß) to promote lung tumourigenesis, we hypothesized that inhibition of IKKß would reduce stemness, migration and invasion of KRAS-mutant human lung cancer cells. We show that KRAS-driven lung tumoursphere-derived cells exhibit stemness features and increased IKKß kinase activity. IKKß targeting by different approaches reduces the expression of stemness-associated genes, tumoursphere formation, and self-renewal, and preferentially impairs the proliferation of KRAS-driven lung tumoursphere-derived cells. Moreover, we show that IKKß targeting reduces tumour cell migration and invasion, potentially by regulating both expression and activity of matrix metalloproteinase 2 (MMP2). In conclusion, our results indicate that IKKß is an important mediator of KRAS-induced stemness and invasive features in lung cancer, and, therefore, might constitute a promising strategy to lower recurrence rates, reduce metastatic dissemination, and improve survival of lung cancer patients with KRAS-driven disease.

Aurora A kinase and its activator TPX2 are potential therapeutic targets in KRAS-induced pancreatic cancer.

PURPOSE: Oncogenic KRAS mutations are found in over 90% of pancreatic ductal adenocarcinomas (PDACs). As yet, however, no effective therapies are available for KRAS-induced malignancies. Therefore, research aimed at the identification of KRAS targets with therapeutic potential is warranted. Our goal was to investigate Aurora A (AURKA) and targeting protein for Xklp2 (TPX2) as potential therapeutic targets in PDAC. METHODS: AURKA and TPX2 expression was assessed using RNAseq and qRT-PCR in PDAC patient samples and matched non-tumor pancreatic tissues. Publicly available PDAC datasets were used to investigate associations of AURKA and TPX2 expression levels with patient survival and the presence of KRAS mutations. Next, we used an Aurora kinase inhibitor, or KRAS, AURKA and TPX2 targeting using RNA interference in KRAS-mutant PDAC cells and, subsequently, analyzed their clonogenic and anchorage-independent growth and migration. RESULTS: We found that relative to matched non-tumor tissues, PDAC tumors displayed significantly higher expression levels of AURKA and TPX2. In addition, we found that AURKA and TPX2 were co-expressed in PDAC datasets, and that high expression levels of AURKA and TPX2 were associated with a shorter patient survival and with the presence of oncogenic KRAS mutations. In addition, we found that siRNA-mediated KRAS targeting in KRAS-mutant PDAC cells reduced AURKA and TPX2 expression. Furthermore, targeting AURKA or TPX2 in KRAS-mutant PDAC cells reduced their clonogenic and anchorage-independent growth, as well their migration. CONCLUSIONS: From our data we conclude that AURKA and TPX2 may act as KRAS biomarkers in PDAC that can predict a worse prognosis, and that AURKA or TPX2 targeting in PDAC cells may reduce their transformed phenotype. These results indicate that AURKA and TPX2 may serve as promising targets to be explored for KRAS-mutant PDAC therapy.

IKKß targeting reduces KRAS-induced lung cancer angiogenesis in vitro and in vivo: A potential anti-angiogenic therapeutic target.

OBJECTIVES: The ability of tumor cells to drive angiogenesis is an important cancer hallmark that positively correlates with metastatic potential and poor prognosis. Therefore, targeting angiogenesis is a rational therapeutic approach and dissecting proangiogenic pathways is important, particularly for malignancies driven by oncogenic KRAS, which are widespread and lack effective targeted therapies. Based on published studies showing that oncogenic RAS promotes angiogenesis by upregulating the proangiogenic NF-κB target genes IL-8 and VEGF, that NF-κB activation by KRAS requires the IKKß kinase, and that targeting IKKß reduces KRAS-induced lung tumor growth in vivo, but has limited effects on cell growth in vitro, we hypothesized that IKKß targeting would reduce lung tumor growth by inhibiting KRAS-induced angiogenesis. MATERIALS AND METHODS: To test this hypothesis, we targeted IKKß in KRAS-mutant lung cancer cell lines either by siRNA-mediated transfection or by treatment with Compound A (CmpdA), a highly specific IKKß inhibitor, and used in vitro and in vivo assays to evaluate angiogenesis. RESULTS AND CONCLUSIONS: Both pharmacological and siRNA-mediated IKKß targeting in lung cells reduced expression and secretion of NF-κB-regulated proangiogenic factors IL-8 and VEGF. Moreover, conditioned media from IKKß-targeted lung cells reduced human umbilical vein endothelial cell (HUVEC) migration, invasion and tube formation in vitro. Furthermore, siRNA-mediated IKKß inhibition reduced xenograft tumor growth and vascularity in vivo. Finally, IKKß inhibition also affects endothelial cell function in a cancer-independent manner, as IKKß inhibition reduced pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Taken together, these results provide a novel mechanistic understanding of how the IKKß pathway affects human lung tumorigenesis, indicating that IKKß promotes KRAS-induced angiogenesis both by cancer cell-intrinsic and cancer cell-independent mechanisms, which strongly suggests IKKß inhibition as a promising antiangiogenic approach to be explored for KRAS-induced lung cancer therapy.

Aurora kinase targeting in lung cancer reduces KRAS-induced transformation.

BACKGROUND: Activating mutations in KRAS are prevalent in lung cancer and have been causally linked to the oncogenic process. However, therapies targeted to oncogenic RAS have been ineffective to date and identification of KRAS targets that impinge on the oncogenic phenotype is warranted. Based on published studies showing that mitotic kinases Aurora A (AURKA) and B (AURKB) cooperate with oncogenic RAS to promote malignant transformation and that AURKA phosphorylates RAS effector pathway components, the aim of this study was to investigate whether AURKA and AURKB are KRAS targets in lung cancer and whether targeting these kinases might be therapeutically beneficial. METHODS: In order to determine whether oncogenic KRAS induces Aurora kinase expression, we used qPCR and western blotting in three different lung cell-based models of gain- or loss-of-function of KRAS. In order to determine the functional role of these kinases in KRAS-induced transformation, we generated KRAS-positive A549 and H358 cells with stable and inducible shRNA-mediated knockdown of AURKA or AURKB and evaluated transformation in vitro and tumor growth in vivo. In order to validate AURKA and/or AURKB as therapeutically relevant KRAS targets in lung cancer, we treated A549 and H358 cells, as well as two different lung cell based models of gain-of-function of KRAS with a dual Aurora kinase inhibitor and performed functional in vitro assays. RESULTS: We determined that KRAS positively regulates AURKA and AURKB expression. Furthermore, in KRAS-positive H358 and A549 cell lines, inducible knockdown of AURKA or AURKB, as well as treatment with a dual AURKA/AURKB inhibitor, decreased growth, viability, proliferation, transformation, and induced apoptosis in vitro. In addition, inducible shRNA-mediated knockdown of AURKA in A549 cells decreased tumor growth in vivo. More importantly, dual pharmacological inhibiton of AURKA and AURKB reduced growth, viability, transformation, and induced apoptosis in vitro in an oncogenic KRAS-dependent manner, indicating that Aurora kinase inhibition therapy can specifically target KRAS-transformed cells. CONCLUSIONS: Our results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy.

Characterisation of a new splice variant of MASK-BP3(ARF) and MASK human genes, and their expression patterns during haematopoietic cell differentiation.

In this study we report the characterisation of a new splice variant, here denominated splice variant 4 (accession number AF258557) of the human Multiple Ankyrin repeats Single KH domain (hMASK) (accession number AF521882) and the hMASK-4E-Binding Protein 3 Alternative Reading Frame (hMASK-BP3(ARF)) (accession number AF521883), containing a number of ANK-repeat motifs. Ankyrin (ANK) repeat-containing proteins carry out a wide variety of biological activities and are involved in processes, such as cell differentiation and transcriptional regulation. The present study reports the computer analysis of these splice variant cDNAs and their broad mRNA expression in different normal human tissues and cancer cell lines. An upregulation of the splice variant mRNAs expression was observed after HL-60 and erythroblast differentiation. The upregulation of splice variant 4 mRNA was considerably higher than those of the other variants, during erythroid differentiation.

Human leukocyte formin: a novel protein expressed in lymphoid malignancies and associated with Akt.

The very large family of Formin proteins is involved in processes such as morphogenesis, embryonic differentiation, cell polarity, and cytokinesis. A novel human gene from the Formin family, denominated human leukocyte formin gene, was cloned. The cDNA of the gene was determined to be 3959bp long with an open reading frame of 3302bp and computational analysis located this gene on chromosome 17, suggesting that it is composed of 27 exons. Northern blot analysis revealed a restricted expression of mRNA in the thymus, spleen, and peripheral blood leukocytes in normal human tissues. Western blot analysis demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies; cancer cell lines and peripheral blood leukocyte from chronic lymphocytic leukemia (CLL) patients. Furthermore, the human leukocyte formin protein was observed to associate with Akt, a critical survival regulator in many different cell types.

ARHGAP10, a novel human gene coding for a potentially cytoskeletal Rho-GTPase activating protein.

Rho-GTPase activating proteins (Rho-GAPs) are negative regulators of Rho-GTPase signaling pathways related to actin cytoskeleton dynamics, cell proliferation, and differentiation. We have identified a novel human gene, termed ARHGAP10, that codes for a 1957-aminoacid Rho-GAP, containing a PDZ, a PH, and a Rho-GAP domain. The cDNA is 7118 bp long and has an open reading frame of 5874 bp. A computational analysis located this gene on chromosome 10 band 10p12.32 suggesting that it is composed of 25 exons. Northern analysis revealed that it is widely expressed, with high levels in brain and muscle. Real-time quantitative PCR analysis confirmed an increase in ARHGAP10 expression during differentiation of HL-60 cells with all-trans-retinoic acid and hematopoietic stem cells with erythropoietin, suggesting that this gene could play a role in normal hematopoiesis. The fact that this gene is highly expressed in muscle and brain, which are highly differentiated tissues, further supports the hypothesis that ARHGAP10 is important for cell differentiation.