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Regulation of Autophagy by VEGF-C axis in cancer

ABSTRACT: A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. In order to develop new treatment modalities or improve the efficacy of current ones it is important to understand the molecular mechanisms that promote therapy-resistance to cancer cells. One pathway that has been demonstrated to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from necrosis. Effective targeting of this pathway could lead to the development of new therapies. In our studies, we found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which is essential for the survival of cancer cells following chemotherapy treatment. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1 that have previously been suggested to participate in autophagy and vesicular trafficking. The upregulation of WDFY-1 upon depleted level of VEGF-C contributed to cytotoxic drug-mediated cell death. Altogether, these data suggest a link between VEGF-C/neuropilin-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress.

ORGANISM(S): Homo sapiens

PROVIDER: GSE36085 | GEO | 2012/04/01

SECONDARY ACCESSION(S): PRJNA151735

REPOSITORIES: GEO

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Project description:A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. In order to develop new treatment modalities or improve the efficacy of current ones it is important to understand the molecular mechanisms that promote therapy-resistance to cancer cells. One pathway that has been demonstrated to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from necrosis. Effective targeting of this pathway could lead to the development of new therapies. In our studies, we found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which is essential for the survival of cancer cells following chemotherapy treatment. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1 that have previously been suggested to participate in autophagy and vesicular trafficking. The upregulation of WDFY-1 upon depleted level of VEGF-C contributed to cytotoxic drug-mediated cell death. Altogether, these data suggest a link between VEGF-C/neuropilin-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress. Human prostate cancer cell lines PC3, Du145 and pancreatic cancer cell line CaPan1 were cultured at 37οC either in RPMI 1640 with L-glutamine or in DMEM media with 10% fetal bovine serum and supplemented with penicillin/streptomycin. PC3 stable transfected cell line was growing in presence of 1µg/ml puromycin selection pressure. Cells were transfected with SmartPool cocktail siRNA for NRP-2, VEGF-C, LAMP-2, and WDFY-1, using DharmaFECT 1-4. siRNA transfection was allowed to proceed 72 h before collection of whole-cell extract or total RNA.To identify potential pathways involved in this VEGF-C-mediated cell survival, we performed a microarray study comparing cells depleted in either VEGF-C or NRP-2 using SmartPool siRNA to PC-3 cells treated with scrambled siRNA. Upon comparison of the two data sets, we found 34 gene-tags that were commonly up- or down-regulated in both NRP-2- and VEGF-C-depleted cells.

Project description:Background: Neuropilin-1 (NRP-1) is a non-tyrosine kinase glycoprotein receptor that elicits multiple functions depending on the ligand bound. It is known to promote cancer progression however; transcriptome-wide changes triggered by NRP-1 are not defined. In this study we aimed to identify novel molecules associated with NRP-1 expression using a global transcriptomic approach in a recombinant breast cancer cell model. Methods: NRP-1 was stably overexpressed in the BT-474 breast cancer cell line (BT-474 NRP-1) and subjected to next generation RNA sequencing using the Illumina HiSeq 4000 sequencing system. Two replicates each of the control BT-474 and BT474-NRP1 were utilized for sequencing. The count per million (CPM) method was utilized for filtering low counts/noise by NOISeq. The clean reads were mapped to reference genome using HISAT/ Bowtie2 tool. The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) method was utilized to calculate the expression levels. False Detection Rate (FDR) ≤ 0.001, absolute value of Log2 ratio ≥ 2 and consistency between the two replicates were used as the default threshold to identify significant DEGs. Results: A total of 22,655,647 clean reads from 22,914,684 raw sequencing reads were generated upon sequencing. 22 upregulated and 61 downregulated genes were identified. Among the DEGs, 11 upregulated and 11 downregulated genes were shortlisted based on their known roles in cancer and/or EMT for confirmation with real-time qPCR. 2 upregulated genes TNC and TARP and 5 downregulated genes ACE, APOD, DDIT3, P2RX6 and ATF3 indicated consistently differential expression with qPCR. Conclusion: We report for the first time the global transcriptome-wide changes elicited by NRP-1 overexpression in a breast cancer cell model. Our study identified the novel association between several candidate molecules such as TNC, a known oncogene, and NRP-1 expression that may controbute to the tumourigenic role of NRP-1 in breast cancer.

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Regulation of Autophagy by VEGF-C axis in cancer

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