Search

Supplementary Figure Legends 1-5 from MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

Supplementary Methods and Materials from MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

Supplementary Figures 1-5 from MicroRNA Replacement Therapy for miR-145 and miR-33a Is Efficacious in a Model of Colon Carcinoma

Simple Summary Glioblastoma is the most common primary brain cancer in adults. One reason for the development and malignancy of this tumor is the misregulation of certain cellular proteins. The oncoprotein STAT3 that is frequently overactive in glioblastoma cells is associated with more aggressive disease and decreased patient survival. Autophagy is a form of cellular self digestion that normally maintains cell integrity and provides nutrients and basic building blocks required for growth. While glioblastoma is known to be particularly resistant to conventional therapies, recent research has suggested that these tumors are more sensitive to excessive overactivation of autophagy, leading to autophagy-dependent tumor cell death. Here, we show a hitherto unknown role of STAT3 in sensitizing glioblastoma cells to excessive autophagy induced with the repurposed drug pimozide. These findings provide the basis for future research aimed at determining whether STAT3 can serve as a predictor for autophagy-proficient tumors and further support the notion of overactivating autophagy for cancer therapy. Abstract Glioblastoma (GBM) is a devastating disease and the most common primary brain malignancy of adults with a median survival barely exceeding one year. Recent findings suggest that the antipsychotic drug pimozide triggers an autophagy-dependent, lysosomal type of cell death in GBM cells with possible implications for GBM therapy. One oncoprotein that is often overactivated in these tumors and associated with a particularly dismal prognosis is Signal Transducer and Activator of Transcription 3 (STAT3). Here, we used isogenic human and murine GBM knockout cell lines, advanced fluorescence microscopy, transcriptomic analysis and FACS-based assessment of cell viability to show that STAT3 has an underappreciated, context-dependent role in drug-induced cell death. Specifically, we demonstrate that depletion of STAT3 significantly enhances cell survival after treatment with Pimozide, suggesting that STAT3 confers a particular vulnerability to GBM. Furthermore, we show that active STAT3 has no major influence on the early steps of the autophagy pathway, but exacerbates drug-induced lysosomal membrane permeabilization (LMP) and release of cathepsins into the cytosol. Collectively, our findings support the concept of exploiting the pro-death functions of autophagy and LMP for GBM therapy and to further determine whether STAT3 can be employed as a treatment predictor for highly apoptosis-resistant, but autophagy-proficient cancers.

Purpose Increased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1. Methods CRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU). Results We found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU. Conclusions Cells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

Pancreatic cancer is among the most detrimental tumors, with novel treatment options urgently needed. The pathological downregulation of a miRNA in tumors can lead to the overexpression of oncogenes, thus suggesting miRNA replacement as novel strategy in cancer therapy. While the role of miR24 in cancer, including pancreatic carcinoma, has been described as ambiguous, it may hold great promise and deserves further studies. Here, we comprehensively analyze the effects of miR24–3p replacement in a set of pancreatic carcinoma cell lines. Transfection of miR24–3p mimics leads to profound cell inhibition in various 2D and 3D cell assays, based on the induction of apoptosis, autophagy and ROS. Comprehensive analyses of miR24–3p effects on the molecular level reveal the transcriptional regulation of several important oncogenes and oncogenic pathways. Based on these findings, miRNA replacement therapy was preclinically explored by treating tumor xenograft-bearing mice with miR24–3p mimics formulated in polymeric nanoparticles. The obtained tumor inhibition was associated with the induction of apoptosis and necrosis. Taken together, we identify miR24–3p as powerful tumor-inhibitory miRNA for replacement therapy, and describe a complex network of oncogenic pathways affected by miR24.

Overcoming apoptosis resistance is one major issue in glioblastoma (GB) therapies. Accumulating evidence indicates that resistance to apoptosis in GB is mediated via upregulation of pro-survival BCL2-family members. The synthetic BH3-mimetic ABT-737 effectively targets BCL2, BCL2 like 1 and BCL2 like 2 but still barely affects cell survival which is presumably due to its inability to inhibit myeloid cell leukemia 1 (MCL1). The constitutively active serine/threonine kinase proviral integration site for moloney murine leukemia virus 1 (PIM1) was recently found to be overexpressed in GB patient samples and to maintain cell survival in these tumors. For different GB cell lines, Western Blot, mitochondrial fractionation, fluorescence microscopy, effector caspase assays, flow cytometry, and an adult organotypic brain slice transplantation model were used to investigate the putative PIM1/MCL1 signaling axis regarding potential synergistic effects with ABT-737. We demonstrate that combination of the PIM1 inhibitor SGI-1776 or the pan-PIM kinase inhibitor AZD1208 with ABT-737 strongly sensitizes GB cells to apoptosis. Unexpectedly, this effect was found to be MCL1-independent, but could be partially blocked by caspase 8 (CASP8) inhibition. Remarkably, the analysis of autophagy markers in combination with the observation of massive accumulation and hampered degradation of autophagosomes suggests a completely novel function of PIM1 as a late stage autophagy regulator, maintaining the autophagic flux at the level of autophagosome/lysosome fusion. Our data indicate that PIM1 inhibition and ABT-737 synergistically induce apoptosis in an MCL1-independent but CASP8-dependent manner in GB. They also identify PIM1 as a suitable target for overcoming apoptosis resistance in GB.

Liver cancer is the fourth leading cause of cancer‑related mortality worldwide with limited therapeutic options. Thus, novel treatment strategies are urgently required. While the oncogenic kinase, proviral integration site for Moloney murine leukemia virus 2 (PIM2), has been shown to be overexpressed in liver cancer, little is known about the role of PIM2 in this tumor entity. In this study, we explored the functional relevance and therapeutic potential of PIM2 in liver cancer. Using PIM2‑specific siRNAs, we examined the effects of PIM2 knockdown on proliferation (WST‑1 assays and spheroid assays), 3D‑colony formation and colony spread, apoptosis (flow cytometry and caspase 3/caspase 7 activity), as well as cell cycle progression (flow cytometry, RT‑qPCR and western blot analysis) in the two liver cancer cell lines, HepG2 and Huh‑7. In subcutaneous liver cancer xenografts, we assessed the effects of PIM2 knockdown on tumor growth via the systemic delivery of polyethylenimine (PEI)‑complexed siRNA. The knockdown of PIM2 resulted in potent anti‑proliferative effects in cells grown on plastic dishes, as well as in spheroids. This was due to G0/G1 cell cycle blockade and the subsequent downregulation of genes related to the S phase as well as the G2/M phase of the cell cycle, whereas the apoptotic rates remained unaltered. Furthermore, colony formation and colony spread were markedly inhibited by PIM2 knockdown. Notably, we found that HepG2 cells were more sensitive to PIM2 knockdown than the Huh‑7 cells. In vivo, the therapeutic nanoparticle‑mediated delivery of PIM2 siRNA led to profound anti‑tumor effects in a liver cancer xenograft mouse model. On the whole, the findings of this study underscore the oncogenic role of PIM2 and emphasize the potential of targeted therapies based on the specific inhibition of PIM2 in liver cancer.

In the original article, the title of the article is “Restoration of MARCK enhances chemosensitivity in cancer”. The authors would like to change the article title to “Restoration of MARCKS enhances chemosensitivity in cancer” by adding a letter “S” to the word MARCK.

Introduction Hepatocellular Carcinoma (HCC) is the sixth most frequent cancer and the second leading cause of cancer related death worldwide, with a poor five year survival and increasing incidence in the Western World. The family of oncogenic serine/threonine kinases comprises three members: Pim1, Pim2 and Pim3. While Pim1 has been the major focus in oncological research and therapeutic exploration so far, Pim2 is overexpressed in HCC as well and has been suggested to be relevant in HCC tumorigenesis. Material and methods siRNA-mediated knockdown of Pim2 cells was determined on (mRNA) and protein level (Western blot). Biological effects of Pim2 knockdown in HCC were explored by investigating cell proliferation (WST-1-assay), cell cycle distribution (flow cytometry), apoptosis (flow cytometry, Caspase activity-assay), and colony formation (clonogenic assay). Changes in downstream signalling pathways leading to the observed biological effects after Pim2 knockdown were explored on mRNA as well as on protein level. The therapeutic potential of a Pim2 was assessed in an in vivo s.c. xenograft mouse model using polymeric nanoparticles based on polyethyleneimine (PEI) as siRNA-delivery platform for systemic treatment. Results and discussions Knockdown of Pim2 using siRNAs lead to strong reduction in Pim2 mRNA and protein levels. This resulted in various cellular effects, including the inhibition of proliferation, increased apoptosis, as well as altered cell cycle distribution and reduced colony formation. On the molecular level, Pim2 knockdown led to changed expression and/or phosphorylation of key proteins involved in the regulation of cell cycle (cyclin B1) and apoptosis (Survivin, Bad). In the in vivo xenograft mouse model, systemic delivery of PEI/siPim2 nanoparticles resulted in profound tumor-inhibitory effects. Conclusion Our results highlight the relevance of the oncogenic serine/threonine kinase Pim2 in HCC. Counteracting its overexpression by siRNA therapeutics, or specifically inhibiting Pim2 on the protein level, might represent novel treatment strategies in HCC therapy.

Introduction Pim-Kinases (Pim - proviral integration site for moloney murine leukemia virus; Pim-1, -2, -3) are a family of serine/threonine kinases with oncogenic potential. They are overexpressed in various solid tumors including hepatocellular carcinoma (HCC) and have been found responsible for malignant transformation, induction of cell proliferation and inhibition of apoptosis. While the main focus has been on Pim-1 so far, the other Pim kinases, esp. Pim-2, have not been explored in detail. Objectives The aim of this study was to compare the tumor-inhibitory effects of the single and combined of Pim family members in HCC, for evaluating their individual contributions and potential as therapeutic target. To this end, RNA interference (RNAi) for specific gene knockdown was employed. Materials and methods Different HCC cell lines were transfected with small interfering RNAs (siRNAs) for inducing RNAi and transient target gene downregulation. Various cellular and molecular effects were analysed. Results Transient siRNA transfection led to robust and specific knockdown of the selected Pim kinase(s) on mRNA and protein level. This resulted in anti-proliferative and pro-apoptotic effects. Most profound consequences on cell growth, cell cycle and apoptosis were observed upon Pim-2 knockdown, with little additive effects upon combined inhibition. These cellular changes were paralleled by alterations of various genes involved in cell cycle, cell survival, EMT and other important cellular processes. Conclusion In hepatocellular carcinoma, Pim-2 represents a functionally relevant oncogene. We demonstrate its higher potential as compared to the other Pim family members, and analyze various consequences of its inhibition or knockdown on the cellular and molecular level. Taken together, this identifies Pim-2 as a potentially relevant target gene for therapeutic intervention. Citation Format: Ulrike Weirauch, Pia Kürz, Achim M. Aigner. Comparison between knockdown of Pim-1, Pim-2 and Pim-3 identifies Pim-2 as the most relevant Pim oncogene in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4125. doi:10.1158/1538-7445.AM2017-4125

RNA interference (RNAi) is a naturally occurring, powerful mechanism for gene silencing, based on the cleavage of a given target mRNA. It relies on small interfering RNAs (siRNAs) in the cell. Being similar in structure, microRNAs (miRNAs) are important regulators of gene expression which mainly act by blocking mRNA translation. In cancer, certain miRNAs have been found to be pathologically downregulated. The therapeutic application of siRNAs or miRNAs for the induction of RNAi or miRNA replacement, respectively, relies on their efficient delivery through a non-viral formulation. Complexation of siRNAs/miRNAs in polymeric nanoparticles based on polyethylenimines (PEIs) offers protection against degradation, delivery to the target site, cellular uptake, and intracellular release. This chapter provides protocols for therapeutic gene silencing and miRNA replacement therapy, based on PEI complexes for in vitro and in vivo use.

U1 small nuclear interference (U1i) has recently been described as a novel gene silencing mechanism. U1i employs short oligonucleotides, so-called U1 adaptors, for specific gene knockdown, expanding the field of current silencing strategies that are primarily based on RNA interference (RNAi) or antisense. Despite the potential of U1 adaptors as therapeutic agents, their in vivo application has not yet been studied. Here we explore U1i by analyzing U1 adaptor-mediated silencing of the oncogene Pim-1 in glioblastoma cells. We have generated Pim-1-specific U1 adaptors comprising DNA, locked nucleic acids (LNA), and 2′-O-Methyl RNA and demonstrate their ability to induce a Pim-1 knockdown, leading to antiproliferative and pro-apoptotic effects. For the therapeutic in vivo application of U1 adaptors, we establish their complexation with branched low molecular weight polyethylenimine (PEI). Upon injection of nanoscale PEI/adaptor complexes into subcutaneous glioblastoma xenografts in mice, we observed the knockdown of Pim-1 that resulted in the suppression of tumor growth. The absence of hepatotoxicity and immune stimulation also demonstrates the biocompatibility of PEI/adaptor complexes. We conclude that U1i represents an alternative to RNAi for the therapeutic silencing of pathologically upregulated genes and demonstrate the functional relevance of Pim-1 oncogene knockdown in glioblastoma. We furthermore introduce nanoscale PEI/adaptor complexes as efficient and safe for in vivo application, thus offering novel therapeutic approaches based on U1i-mediated gene knockdown.

RNA interference (RNAi) is a naturally occurring, powerful mechanism for gene silencing, based on the cleavage of a given target mRNA. It relies on small interfering RNAs (siRNAs) in the cell. Being similar in structure, microRNAs (miRNAs) are important regulators of gene expression which mainly act by blocking mRNA translation. In cancer, certain miRNAs have been found to be pathologically downregulated. The therapeutic application of siRNAs or miRNAs for the induction of RNAi or miRNA replacement, respectively, relies on their efficient delivery through a non-viral formulation. Complexation of siRNAs/miRNAs in polymeric nanoparticles based on polyethylenimines (PEIs) offers protection against degradation, delivery to the target site, cellular uptake, and intracellular release. This chapter provides protocols for therapeutic gene silencing and miRNA replacement therapy, based on PEI complexes for in vitro and in vivo use.

Antisense inhibition of oncogenic or other disease-related miRNAs and miRNA families in vivo may provide novel therapeutic strategies. However, this approach relies on the development of potent miRNA inhibitors and their efficient delivery into cells. Here, we introduce short seed-directed LNA oligonucleotides (12- or 14-mer antiseeds) with a phosphodiester backbone (PO) for efficient miRNA inhibition. We have analyzed such LNA (PO) antiseeds using a let-7a-controlled luciferase reporter assay and identified them as active miRNA inhibitors in vitro. Moreover, LNA (PO) 14-mer antiseeds against ongogenic miR-17–5p and miR-20a derepress endogenous p21 expression more persistently than corresponding miRNA hairpin inhibitors, which are often used to inhibit miRNA function. Further analysis of the antiseed-mediated derepression of p21 in luciferase reporter constructs - containing the 3′-UTR of p21 and harboring two binding sites for miRNAs of the miR-106b family - provided evidence that the LNA antiseeds inhibit miRNA families while hairpin inhibitors act in a miRNA-specific manner. The derepression caused by LNA antiseeds is specific, as demonstrated via seed mutagenesis of the miR-106b target sites. Importantly, we show functional delivery of LNA (PO) 14-mer antiseeds into cells upon complexation with polyethylenimine (PEI F25-LMW), which leads to the formation of polymeric nanoparticles. In contrast, attempts to deliver a functional seed-directed tiny LNA 8-mer with a phosphorothioate backbone (PS) by formulation with PEI F25-LMW remained unsuccessful. In conclusion, LNA (PO) 14-mer antiseeds are attractive miRNA inhibitors, and their PEI-based delivery may represent a promising new strategy for therapeutic applications.

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR-33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3′-untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.

MicroRNAs control the expression of various genes, and several miRNAs are aberrantly expressed in tumors. MiRNA-145 is markedly downregulated in various cancers including colon carcinoma, and in vitro studies have established its pro-apoptotic and anti-proliferative role. MiR-33a has only recently been connected to cancer through its capacity to downregulate the oncogenic kinase Pim-1. Despite the functional relevance of miRNAs suppressed in tumors, only a few studies have explored their therapeutic application. This miRNA replacement therapy is largely hampered by the lack of powerful non-viral delivery tools for their in vivo administration. In this paper, we establish, for the first time, miRNA replacement therapy by polyethylenimine (PEI)-mediated delivery of unmodified miRNAs. After systemic or local application of low molecular weight PEI/miRNA complexes, intact miRNA molecules are delivered into tumors, resulting in profound anti-tumor effects in s.c. colon carcinoma xenograft mouse models. Tumor inhibition is based on miR-145-mediated reduced proliferation, increased apoptosis and the concomitant repression of c-Myc and ERK5, which we identify for the first time as being regulated by miR-145. Furthermore, we introduce the systemic injection of PEI-complexed miR-33a as a novel therapeutic principle aiming at the in vivo downregulation of the oncogenic kinase Pim-1. Anti-tumor effects of the treatment with PEI/miRNA complexes are comparable to PEI/siRNA-mediated Pim-1 knockdown in vivo. Taken together, this is the first study to establish chemically unmodified miRNAs, complexed with PEI, as an efficient and biocompatible strategy for miRNA replacement therapy, and to introduce PEI/miR-145 and PEI/miR-33a complexes as anti-tumor compounds in colon carcinoma.

The Argonaute proteins play essential roles in development and cellular metabolism in many organisms, including plants, flies, worms, and mammals. Whereas in organisms such as Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute mutant strains allowed the study of their biological functions, in mammals the application of this approach is limited by its difficulty and in the specific case of Ago2 gene, by the lethality of such mutation. Hence, in human cells, functional studies of Ago proteins relied on phenotypic suppression using small interfering RNA (siRNA) which involves Ago proteins and the RNA interference mechanism. This bears the danger of undesired or unknown interference effects which may lead to misleading results. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with processes in which Agos are involved. Different functional observations in the use of siRNA versus asONs indicate the relevance of this assumption. We developed asONs specific for the four human Agos (hAgos) and compared their activities with those obtained by siRNA. We confirm that hAgo2 is involved in microRNA (miRNA)- and in siRNA-mediated silencing pathways, while the other hAgos play a role only in miRNA-based gene regulation. Using combinations of asONs we found that the simultaneous down-regulation of hAgo1, hAgo2, and hAgo4 led to the strongest decrease in miRNA activity, indicating a main role of these proteins.

Pim-1 is a constitutively active serine/threonine kinase overexpressed in various tumors. Its role as proto-oncogene is based on several Pim-1 target proteins involved in pivotal cellular processes, and Pim-1 overexpression has been linked to poor prognosis. RNAi-based knockdown approaches were used to inhibit Pim-1 in colon carcinoma cells. We demonstrate anti-proliferative, pro-apoptotic and overall antitumor effects of Pim-1 inhibition. The analysis of the molecular effects of Pim-1 inhibition reveals a complex regulatory network, with therapeutic Pim-1 repression leading to major changes in oncogenic signal transduction with regard to p21Cip1/WAF1, STAT3, JNK, c-Myc and survivin, and in the levels of apoptosis-related proteins Puma, Bax and Bcl-xL. Furthermore, Pim-1 knockdown sensitizes tumor cells towards 5-FU treatment, thereby antagonizing a 5-FU-triggered Pim-1 upregulation. This effect is mediated through decreased miR-15b levels, and our studies identify miR-15b and miR-33a to regulate Pim-1. There is first evidence of Pim-1 overexpression also in glioblastoma, but the functional relevance is so far unknown. U1 small nuclear interference (U1i) has recently been described as novel gene silencing mechanism. It employs short oligonucleotides, so-called U1 adaptors, for specific gene knockdown. We generated Pim-1 specific U1 adaptors and demonstrate their ability to induce Pim-1 knockdown in GBM cells. U1 adaptor transfection leads to anti-proliferative and pro-apoptotic effects. To explore therapeutic in vivo applications, we complexed siRNAs or U1 adaptors with a low molecular weight polyethylenimine (PEI) that mediates protection and cellular internalization. Treatment of tumor xenograft-bearing mice with PEI/siRNA or PEI/adaptor nanoparticles exhibits antitumor effects based on the knockdown of Pim-1, thus offering novel therapeutic strategies. Conclusions: We demonstrate that Pim-1 plays a pivotal role in several tumor-relevant signalling pathways, and establish the functional relevance of Pim-1 in colon carcinoma and in glioblastoma. Our results also substantiate the RNAi-mediated Pim-1 knockdown based on polymeric PEI/siRNA nanoparticles as a promising therapeutic approach, and we show that U1i represents an alternative to RNAi for the therapeutic knockdown of pathologically upregulated genes. Citation Format: Ulrike Weirauch, Maren Thomas, Arnold Grünweller, Roland K. Hartmann, Achim M. Aigner. RNAi- and U1 small nuclear interference (U1i)-mediated gene knockdown reveals the functional relevance of Pim-1 kinase in colon carcinoma and glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2187. doi:10.1158/1538-7445.AM2013-2187

Various miRNAs are aberrantly expressed in cancer. In colon carcinoma, decreased levels of the pro-apoptotic and anti-proliferative miRNA-145 are observed, while the role of miR-33a has not been analysed yet. In this study, we demonstrate the tumor-inhibitory role of miR-33a and newly identify the proto-oncogenic kinase Pim-1 as its direct target. Comparably to siRNA-mediated knockdown of Pim-1, miR-33a reduces Pim-1 expression and thus inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression. Most recent in vitro and in vivo data from our group further establish the so far unknown functional relevance of Pim-1 in colon carcinoma and identify Pim-1 as attractive target gene. Among others, RNAi-mediated Pim-1 knockdown reduces tumor growth, induces apoptosis and leads to major changes in oncogenic signal transduction, including the inhibition of STAT3. The therapeutic application of miRNAs strongly relies on the development of suitable delivery tools, and we introduce polyethylenimine (PEI)-based nanoparticles for systemic or local miRNA administration in vivo. PEIs mediate miRNA protection, delivery to target organs, cellular uptake and intracellular release. MiRNA replacement therapy through systemic or local injection of PEI/miR-145 complexes results in efficient miRNA delivery and in antitumor effects in s.c. colon carcinoma xenograft mouse models. Likewise, tumor growth inhibition is observed upon treatment with PEI-complexed miR-33a. This is due to the miR-33a-mediated downregulation of Pim-1 expression, comparable with the Pim-1 knockdown through PEI/siRNA complexes. Conclusions: (i) The PEI-complexation of miRNAs represents a novel strategy in miRNA replacement therapy, (ii) miR-145 / miR-33a may be promising candidate miRNAs, and (iii) Pim-1 is a newly identified, attractive target gene in colon carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-482. doi:1538-7445.AM2012-LB-482