Mateus Nóbrega Aoki

Association study of CCR5 delta 32 polymorphism among the HLA-DRB1 Caucasian population in Northern Paraná, Brazil.

Chemokines are important determinants of early inflammatory response. The CC chemokine receptor 5 (CCR5) delta 32 variant results in a nonfunctional form of the chemokine receptor and has been implicated in a variety of immune‐mediated diseases. In the present study, polymerase chain reaction (PCR) for genomic deoxyribonucleic acid (DNA) samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 225‐basepair (bp) product from the normal CCR5 allele and a 193‐bp product from the 32 bp deletion allele. Human leukocyte antigen (HLA) class II (DRB1) typing was performed by PCR‐sequence‐specific primer (PCR‐SSP). The aim of this study was to evaluate the association of HLA‐DRB1 and CCR5 genetic polymorphisms. To evaluate the frequency distributions of CCR5 delta 32 polymorphisms in a Brazilian population and their association with allelic distribution of HLA genes, DRB1; a total of 120 Caucasian individuals from northern Paraná, Brazil, were tested. The CCR5/CCR5 genotype was found in 108 individuals (90%) and only one carried the CCR5 delta 32 allele homozygous genotype (0.0238), while 12 (10%) carried the CCR5 delta 32 allele heterozygous genotype. The observed frequency for the CCR5 delta 32 allele was 0.05 in the population studied. The results revealed a CCR5 delta 32 allele occurrence with HLA‐DRB1*01 and DRB1*04 (P<0.05). It is possible that HLA‐DRB1*01 and DRB1*04 alleles could be associated with the delta 32‐bp deletion of CCR5. J. Clin. Lab. Anal. 22:229–233, 2008.

Aurora kinase targeting in lung cancer reduces KRAS-induced transformation

BackgroundActivating 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.MethodsIn 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.ResultsWe 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.ConclusionsOur 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.

Abstract 533: MicroRNA486-5p is a KRas target involved in promoting cell proliferation in lung cancer

KRAS-induced lung cancer is a very common disease, for which there are currently no effective therapies. Direct targeting of KRAS has failed in clinical trials and intense efforts are underway to identify KRAS targets that play a crucial role in oncogenesis. One promising KRAS-regulated pathway that has so far been overlooked is the microRNA pathway. Even though many microRNAs that regulate expression of KRAS are known, microRNAs regulated by oncogenic KRAS remain largely unknown. Our goal was to identify microRNAs regulated by oncogenic KRAS in lung cells that could contribute to the oncogenic phenotype. Due to a reported positive correlation between microRNA486-5p (miR-486-5p) expression and the presence of KRAS mutations in colon cancer specimens, we decided to investigate in lung cells whether KRAS regulates miR-486-5p. For that purpose we used an immortalized human primary lung epithelial cell line (SALEB) and its isogenic KRAS-transformed counterpart (SAKRAS), as well as lung cancer cell lines with either gain-of-function or loss-of-function of KRAS. We found, in all models tested, a positive correlation between expression of oncogenic KRAS and expression of miR-486-5p. We also found a negative correlation between expression of oncogenic KRAS and expression of miR-486-5p targets, including FoxO1, a tumor suppressor. In order to evaluate how miR-486-5p affects Ras-induced oncogenic properties, we transfected miR-486-5p inhibitor oligonucleotides into KRAS-positive lung cancer cell lines H358 and A549. Inhibition of miR-486-5p expression leads to reduced growth in clonogenic assays and reduced MTT viability. This reduction is not associated with increased cell death, but, as measured by 3H-Thymidine incorporation, it is associated with decreased cell proliferation. Interestingly, transfection of miR-486-5p double-stranded RNA mimic oligonucleotides into KRAS negative lung cancer cell line H1703 or into H358 cells with loss of function of KRAS by RNA interference leads to enhanced proliferation and clonogenicity. Taken together, these results indicate, not only that miR-486-5p is a KRAS target in lung cancer, but also that miR-486-5p acts as an oncomiR contributing to KRAS-induced cell proliferation. Further understanding of miR-486-5p targets could uncover novel pathways for KRAS-induced lung cancer therapy design. Citation Format: Mateus N. Aoki, Amanda C. P. Salviatto, Tatiana C. C. Lobo, Ana Claudia O. Carreira, Mari C. Sogayar, Daniela S. Basseres. MicroRNA486-5p is a KRas target involved in promoting cell proliferation in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 533. doi:10.1158/1538-7445.AM2014-533

Abstract 4390: Aurora kinases: potential therapeutic targets in K-Ras-induced lung cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC K-Ras-induced lung cancer is a very common disease, for which there are currently no effective therapies. Intense efforts are underway to identify K-Ras targets that play a crucial role in oncogenesis. We have previously shown that K-Ras-induced lung tumorigenesis is potentiated by the transcription factor NF-κB; and others have shown that Aurora kinases, not only play a significant role in tumorigenesis, but also can activate NF-κB. Therefore, we hypothesized that Aurora A and/or Aurora B are important K-Ras targets in lung cancer. In order to test this hypothesis, we first determined whether oncogenic K-Ras induces Aurora kinase expression. For that purpose, we used three different cell-based models: (1) an immortalized primary lung epithelial cell line and its isogenic K-Ras-transformed counterpart, (2) H1703 lung cancer cell line engineered to express oncogenic K-Ras inducibly, and (3) K-Ras positive lung cancer cell lines H358 and A549 stably expressing inducible shRNAs targeting K-Ras. In all cases, K-Ras expression positively correlated with Aurora A and Aurora B expression. In order to validate Aurora A an B as therapeutically relevant K-Ras targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate Aurora A or B. Inducible shRNA-mediated knockdown of Aurora A or B, as well as treatment with a dual Aurora A and B inhibitor (AI II), decreased growth, viability and migration of K-Ras positive H358 and A549 cell lines. Interestingly, in our primary isogenic model and in our H1703 K-Ras-inducible cell line, AI II reduced viability and induced apoptosis in an oncogenic K-Ras-dependent manner. This suggests that Aurora kinase inhibition therapy can specifically target K-Ras transformed cells. In conclusion, our results support our hypothesis that Aurora kinases are important K-Ras targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for K-Ras-induced lung cancer therapy. Citation Format: Edmilson O. dos Santos, Mateus N. Aoki, Daniela S. Basseres. Aurora kinases: potential therapeutic targets in K-Ras-induced lung cancer. [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 4390. doi:10.1158/1538-7445.AM2013-4390