Molecular Consequences of Silencing Mutant K-<i>ras</i> in Pancreatic Cancer Cells: Justification for K-<i>ras</i>–Directed Therapy

Fleming, Jason B.; Shen, Gang; Holloway, Shane E.; Davis, M. Natalie; Brekken, Rolf A.

doi:10.1158/1541-7786.mcr-04-0206

Cited by 173 publications

(147 citation statements)

References 63 publications

(47 reference statements)

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“…Most studies dealing with K-ras silencing reported that siRNA or shRNA molecules could specifically inhibit mutant K-ras expression while leaving the WT K-ras unaffected although their sequences differed only in one nucleotide. [8][9][10][11]13 In our study, siRNA molecules that targeted K-ras mRNA outside the region with mutated codon reduced the levels of mRNA and protein of K-ras in both LoVo cells with mutated K-ras and in HT29 cells harboring WT K-ras. However, our results demonstrated that in HT29 cells, in which their oncogenic potential is due to other mutations, the effect of silencing K-ras did not reflect on cell survival.…”

Section: Discussionsupporting

confidence: 48%

“…The obtained results are consistent with the results from other in vitro studies performed mostly on pancreatic cell lines (Capan-1, Panc-1, MiaPaca-2 and PC-7), in which therapeutic potential of siRNA/ short hairpin RNA (shRNA) molecules directed against K-ras was also shown. [8][9][10][11][12][13] Furthermore, due to a relatively short half-life of synthetic siRNA molecules, 12,13,29,32 we constructed plasmid DNA that encoded a hairpin type of silencing molecules, that is, miRNA molecules with a sequence corresponding to the most efficient siRNA molecule (pmiRNA-K-ras). Most studies dealing with K-ras silencing reported that siRNA or shRNA molecules could specifically inhibit mutant K-ras expression while leaving the WT K-ras unaffected although their sequences differed only in one nucleotide.…”

Section: Discussionmentioning

confidence: 99%

“…It was shown that properly designed small interfering RNA (siRNA) sequences efficiently inhibited the expression of mutated K-ras, whereas expression of wild-type (WT) K-ras remained unaffected. [8][9][10][11][12] The inhibition of K-ras expression by RNAi was reflected in reduced K-ras mRNA and proteins level, reduced proliferation and migration of cells, increased apoptosis, reduced angiogenic potential and decreased production of vascular endothelial cells. In the experiments, in vivo, on pancreatic tumors, delay in tumor growth was observed.…”

Section: Introductionmentioning

confidence: 99%

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MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

et al. 2010

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Mutations of K-ras have been found in 30-60% of colorectal carcinomas and are believed to be associated with tumor initiation, tumor progression and metastasis formation. Therefore, silencing of mutant K-ras expression has become an attractive therapeutic strategy for colorectal cancer treatment. The aim of our study was to investigate the effect of microRNA (miRNA) molecules directed against K-ras (miRNA-K-ras) on K-ras expression level and the growth of colorectal carcinoma cell line LoVo in vitro and in vivo. In addition, we evaluated electroporation as a gene delivery method for transfection of LoVo cells and tumors with plasmid DNA encoding miRNA-K-ras (pmiRNA-K-ras). Results of our study indicated that miRNAs targeting K-ras efficiently reduced K-ras expression and cell survival after in vitro electrotransfection of LoVo cells with pmiRNA-K-ras. In vivo, electroporation has proven to be a simple and efficient delivery method for local administration of pmiRNA-K-ras molecules into LoVo tumors. This therapy shows pronounced antitumor effectiveness and has no side effects. The obtained results demonstrate that electrogene therapy with miRNA-K-ras molecules can be potential therapeutic strategy for treatment of colorectal cancers harboring K-ras mutations.

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Section: Discussionsupporting

confidence: 48%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

et al. 2010

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“…Previous studies reported that inhibition of KRAS expression led to the inhibition of pancreatic cancer cell growth (12,16). To examine the potential growth-inhibiting effect of siG12D, we measured both cell viability and cell death of Panc1 cells treated with the siG12D LODER.…”

Section: Loder-derived Sig12d Significantly Inhibits Growth Of Pancrementioning

confidence: 99%

Mutant KRAS is a druggable target for pancreatic cancer

Khvalevsky¹,

Gabai²,

Rachmut³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

258

187

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Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge. PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target. We propose an approach to target KRAS effectively in patients using RNA interference. To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER). The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo. The effect of the siG12D LODER on tumor growth was assessed in s.c. and orthotopic mouse models. KRAS silencing effect was further assessed on the KRAS downstream signaling pathway. The LODER-encapsulated siRNA was stable and active in vivo for 155 d. Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial-mesenchymal transition. In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival. We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA. This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target.targeted therapy | gene therapy P ancreatic cancer is an aggressive disease that develops in a relatively symptom-free manner and in most cases, is already advanced at the time of diagnosis (1). It has one of the highest fatality rates of all cancers and is one of the leading causes of cancer-related deaths in the Western world (1, 2). Pancreatic ductal adenocarcinoma (PDA) is the most common pancreatic neoplasm, responsible for 95% of pancreatic cancer cases (3). Genetic alterations in the KRAS signaling pathway are involved in over 90% of pancreatic cancer cases (4-6). KRAS mutations were shown to be an early event in the development of pancreatic cancer (5,7,8).The most common KRAS mutation of the human pancreas adenocarcinoma is a gain-of-function substitution mutation of glycine at codon 12 to aspartate (G12D) (5, 9-11). Moreover, PDA cancer cell growth was shown to be dependent on the activity of the mutated KRAS (5, 11) and accordingly, silencing KRAS has proven effective in controlling pancreatic cell line proliferation (12). Here, we aimed to harness the advantages of siRNA technology as a therapeutic modality for pancreatic cancer.Parenteral controlled drug delivery systems are used to improve and advance the therapeutic effects of drug treatments by providing optimized local drug concentrations over prolonged periods of time, reduction of side effects, and cost reduction (13). A prominent method of controlling the release rate of a drug in a pharmaceutical dosage is to embed the active ag...

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“…The clinical significance of the K-ras gene mutation for effective diagnosis of early pancreatic cancer may exceed imaging and cytology examination (21). Gene targeting and immunotherapy for codon 12 of the K-ras gene may aid in the diagnosis and treatment of pancreatic cancer (22). Research in the biological treatment for tumors has confirmed that DC vaccines have significant value in the treatment of malignant tumors and infectious diseases.…”

Section: Discussionmentioning

confidence: 99%

Induction of CTLs by DCs pulsed with K-ras mutant peptide on the surface of nanoparticles in the treatment of pancreatic cancer

Wang¹

2011

Oncol Rep

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Abstract. the aim of this study was to investigate the role of specific cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) presenting cationic nanoparticles with the K-ras (12-Val) mutant peptide in the killing of different pancreatic cancer cell lines in vivo and in vitro. Peripheral blood DCs were induced by rhGM-CSF and IL-4 and cultured. DCs were sensitized by whole antigen of PANC-1 with expression of K-ras mutant, K-ras mutant peptide (K-ras+peptide) and cationic nanoparticles with K-ras mutant peptide (K-ras+peptide-CNP), respectively. Cell surface markers were measured by flow cytometry. Lymphocyte proliferation was detected by the 3 H-tdr test, and IL-12 and IFN-γ secretion was detected by ELISA. 125 I-UdR was used to measure the killing effect of CTLs. The antitumor activity of CTLs in tumor-bearing nude mouse models prepared with PANC-1 and SW1990 cells was evaluated. Results showed that, compared with K-ras+peptide, low concentrations of K-ras+peptide-CNP were effectively presented by DCs (P<0.05). CTLs induced by DCs pulsed with whole tumor antigen had a significantly greater killing effect (P<0.05) on PANC-1 and SW1990 pancreatic cancer cells compared with K-ras+peptide-and K-ras+peptide-CNP-induced CTLs. CTLs induced by DCs pulsed with K-ras+peptide and K-ras+peptide-CNP had a specific killing effect (P<0.05) on PANC-1 cells and no effect (P>0.05) on SW1990 cells. In conclusion, cationic nanoparticles with the K-ras (12-Val) mutant peptide can be effectively presented by DCs at a low concentration. CTLs induced by K-ras+peptide-CNP had specific killing activity for the pancreatic cancer cell line with the K-ras mutant and significantly inhibited tumor growth and increased the survival time of tumor-bearing nude mice. Although this study confirmed that whole cell antigen induced a good antitumor immune response, the possibility of immune tolerance and autoimmunity which has been previously proven contribute to the difficulty in the application of this DC vaccine.

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Molecular Consequences of Silencing Mutant K-ras in Pancreatic Cancer Cells: Justification for K-ras–Directed Therapy

Cited by 173 publications

References 63 publications

MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

MicroRNAs targeting mutant K-ras by electrotransfer inhibit human colorectal adenocarcinoma cell growth in vitro and in vivo

Mutant KRAS is a druggable target for pancreatic cancer

Induction of CTLs by DCs pulsed with K-ras mutant peptide on the surface of nanoparticles in the treatment of pancreatic cancer

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