SIRNAC PROJECT
SIRNAC - NOVEL siRNA THERAPIES AGAINST METASTATIC COLORECTAL CANCER
Is the project of a new therapy against colorectal cancer metastatic to the liver and aims at reducing significantly the impacts of a disease with an extremely heavy human and economical burden.
The project is co-promoted by Phyzat Biopharmaceuticals, INEB – National Institute of Biomedical Engineering, and IPO Porto Oncology Institute.
The SIRNAC project covers the development phase of a new medicine, following the phase of discovery, successfully finished by Phyzat Biopharmaceuticals, with proof of efficacy in vitro and in vivo of the elected molecules. It comprises the development of the elected molecules, the development of the delivery system and the validation of the clinical and therapeutical relevance, either isolated or sinergistically of the two new targets. This project will allow to finish the development phase of this program of a new therapy against colorectal cancer metastatic to the liver and outlicense said program, with the associated PI, to a pharmaceutical company that will start regulatory trials leading to market approval.
The SIRNAC project has a budget of 917.676,75 €, and is supported by European Union public funding of 729.902,80 € through FEDER. SIRNAC was started in July 2018 and is due to finish in June 2020.
Project results include the following publications:
Downregulation of LAT1 (SLC7A5) and ASCT2 (SLC1A5) in human cancer
A. Oliveira1, C. Carneiro1, P. Serrão2, P. Kennedy1, P. Soares-da-Silva1,2;
1Phyzat Biopharmaceuticals, Porto, Portugal, 2MedInUP – Center for Drug Discovery and Innovative Medicines, Porto, Portugal
Amino acid transporters LAT1 and ASCT2 are highly expressed in cancer cells to support their continuous growth and proliferation and have been suggested as markers of cancer prognosis in different types of cancer (Semin Cancer Biol 15:254-266,2005). The present study examined the effect of LAT1 and ASCT2 gene downregulation by RNA interference on the function of the transporters in human cancer cells. Target sites within LAT1 gene (NM003486) and ASCT2 gene (NM001145144) were selected from the respective human mRNA sequences. The commercial siRNA SI31011000 that targets the human LAT1 mRNA sequence, the SI00079730 that targets the human ASCT2 mRNA sequence, and negative control siRNA commercial sequences NC- SI03650318 and NC-SI03650325 (all from Qiagen) were tested. Cells (human liver hepatocellular carcinoma HepG2, human liver adenocarcinoma SK-HEP-1, human fibrosarcoma HT-1080 and human bladder carcinoma T24) were cultured for 48 h (in RPMI, DMM-lg and DMEM-hg, respectively). LAT1 and ASCT2 mRNA and protein expression, inward transport of [14C]-L-leucine and [14C]-L-alanine, and effects of siRNA anti-LAT1 upon [14C]-L-leucine uptake and siRNA anti- ASCT2 upon [14C]-L-alanine uptake were analyzed. Antibodies raised against LAT1, ASCT2 and GAPDH were used, and images were obtained by scanning at both 700 nm and 800 nm, with an Odyssey Infrared Imaging System (LI-COR Biosciences). For RT-PCR, total RNA was converted to cDNA and qPCR analysis was made in the StepOnePlus instrument (Applied Biosystems). Primers for LAT1, ASCT2 and for the endogenous control gene GAPDH were used. All cell types expressed LAT1 and ASCT2 mRNA and protein. The abundance of LAT1 and ASCT2 mRNA differed among the tumor cell lines, being more intense in HT-1080, followed by SK-HEP-1 and T24 cells. The same trend was not verified for LAT1 protein abundance, with T24 cells expressing more LAT1 protein than the other cell lines. The abundance of ASCT2 protein followed the same trend. The kinetic parameters of [14C]-L-leucine and [14C]-L-alanine uptake (Km in mM; Vmax in nmol/mg protein/min) were determined by non-linear analysis of inhibition curves for L-leucine and L-alanine. Transfection with siRNA against LAT1 or ASCT2 genes, but not the negative control siRNAs, reduced the [14C]-L-leucine and [14C]-L-alanine accumulation in all cancer cell lines. In conclusion, anti-LAT1 siRNA and anti-ASCT2 siRNA decreased [14C]-L-leucine and [14C]-L- alanine uptake, possibly by downregulation of LAT1 and ASCT2 expression and function, respectively.
Part of this work was developed within the SIRNAC project, co-financed by NORTE2020, PT2020 and the European Union.
Downregulation of LAT1 (SLC7A5) and ASCT2 (SLC1A5) and proliferation of human colon cancer HCT-116 cells
C. Carneiro1, A. Oliveira1, P. Serrão2, P. Kennedy1, P. Soares-da-Silva1,2;
1Phyzat Biopharmaceuticals, Porto, Portugal, 2MedInUP – Center for Drug Discovery and Innovative Medicines, Porto, Portugal
Amino acid transporter LAT1 is highly expressed in cancer cells and has been suggested as a marker of rectal cancer prognosis (Anticancer Res 30: 4223-4227,2010). Amino acid transporter ASCT2 is expressed in colorectal adenocarcinomas and patient survival decreased with increased percentage of ASCT2-positive cancer cells (Anticancer Res 22:2555-2557,2002; Mol Imaging Biol 19:421-428,2017). The present study examined the effect of LAT1 and ASCT2 gene downregulation by RNA interference on the expression and function of the transporter and cell proliferation of human colon cancer HTC-116 cells. Commercial siRNAs SI31011000 (Qiagen) and HSS112005 (Invitrogen) targeting human LAT1 mRNA sequences, SI00079730 (Qiagen) targeting the human ASCT2 mRNA sequence, and negative control siRNA commercial sequences NC- SI03650318 and NC-SI03650325 (Qiagen) were tested. Cells were transfected with siRNAs complexed with Lipofectamine 2000® or Injectin®. LAT1 and ASCT2 mRNA expression, protein abundance, inward transport of [14C]-L-leucine and [14C]-L-alanine and the effect of siRNA sequences in cell proliferation were evaluated after 72 h. Anti-LAT1 siRNA sequences tested reduced LAT1 mRNA expression around 50% or more when compared to the corresponding vehicle. LAT1 protein expression was also reduced for at least 50%, and this effect was accompanied with a reduction upon the [14C]-L-leucine accumulation in HCT-116 cells. Moreover, LAT1 downregulation decreased cell proliferation, particularly when siRNA sequence HSS112005 was used. The anti-ASCT2 siRNA SI00079730 reduced ASCT2 mRNA expression to 30% when compared to the corresponding vehicle. ASCT2 protein expression was also reduced by approximately 50%, and this effect was associated with a 40% reduction upon the [14C]-L-alanine accumulation in HCT-116 cells. Moreover, ASCT2 downregulation markedly decreased (~70%) cell proliferation. The negative control siRNAs were devoid of effects on parameters measured. In conclusion, anti-LAT1 and anti-ASCT2 siRNA decreased [14C]-L-leucine and [14C]-L-alanine uptake and cellular proliferation in association with downregulation of LAT1 and ASCT2 expression and function, respectively. The decrease in cell viability and proliferation of cancer cells induced by anti-LAT1 and anti-ASCT2 siRNAs is expected provide a decrease in tumor growth and metastasis potential in colon cancer.
Part of this work was developed within the SIRNAC project, co-financed by NORTE2020, PT2020 and the European Union.