Abstract
To explore a novel strategy in suppressing tumor metastasis, we took the advantage of a recent RNA activation (RNAa) theory and used small double-strand RNA molecules, termed as small activating RNAs (saRNA) that are complimentary to target gene promoter, to enhance transcription of metastasis suppressor gene. The target gene in this study is Dihydro-pyrimidinase-like 3 (DPYSL3, protein name CRMP4), which was identified as a metastatic suppressor in prostate cancers. There are two transcriptional variants of DPYSL3 gene in human genome, of which the variant 2 is the dominant transcript (DPYSL3v2, CRMP4a) but is also significantly down-regulated in primary prostate cancers. A total of 8 saRNAs for DPYSL3v1 and 14 saRNAs for DPYSL3v2 were tested in multiple prostate cancer cell lines. While none of the saRNAs significantly altered DPYSL3v1 expression, 4 saRNAs showed a strong enhancing effect on DPYSL3v2 expression, resulting in reduced cell mobility in vitro. To achieve a prostate cancer-specific delivery for in vivo testing, we conjugated the most potent saV2-9 RNA molecule with the prostate-specific membrane antigen (PSMA)-targeting aptamer A10-3.2. The conjugates successful increased DPYSL3v2 gene expression in PSMA-positive but not PSMA-negative prostate cancer cells. In nude mice bearing orthotopic xenograft of prostate cancer, a 10-day consecutive treatment with the saV2-9 conjugates significantly suppress distal metastasis compared to the control saRNAs. Analysis of xenograft tissues revealed that DPYSL3v2 expression was largely increased in saV2-9 conjugate-treated group compared to the control group. In conclusion, DPYSL3v2 promoter-targeted saRNA molecules might be used as an adjunctive therapy to suppress prostate cancer metastasis.
Highlights
Metastasis is the major cause of mortality from prostate cancer [1, 2] and systemic metastasis often occurs following local therapy failure in high-risk patients, including locally advanced or high grade (Gleason sum score ≥ 8) tumors [3, 4]
The isoform-1 has 2055 nt in cDNA nucleotide sequence while isoform-2 is 1713 nt. These isoforms are translated to proteins of CRMP4b (DPYSL3v1, 684 aa, 75 KD) and CRMP4a (DPYSL3v2, 570 aa, 64 KD)
As a proof-of-concept project, this study demonstrated that the small activating RNAs (saRNA) approach is a potent method to enhance the expression of tumor metastasis suppressor Dihydropyrimidinase-related protein 3 (DPYSL3) gene, which depends on the sequence specificity of the target gene promoter
Summary
Metastasis is the major cause of mortality from prostate cancer [1, 2] and systemic metastasis often occurs following local therapy failure in high-risk patients, including locally advanced (positive surgical margin) or high grade (Gleason sum score ≥ 8) tumors [3, 4]. Prostate cancers often migrate to extra-prostatic tissue and local lymph nodes before distant organ metastasis. Suppressing cancer cell invasion at a very early stage would prevent the development of distal metastatic disease and slow down www.impactjournals.com/oncotarget disease progression, improving patient quality of life for prostate cancers. In a search for metastasis-associated proteins using proteomics approach, we previously identified CRMP4 protein as a tumor metastasis suppressor in prostate cancers [14]. Once overexpressed or up-regulated in prostate cancer cells, CRMP4 protein suppressed cell motility or invasion in vitro and reduced tumor metastasis in mouse xenograft models [14,15,16]. Upregulating DPYSL3 gene expression in prostate cancer is expected to suppress tumor metastasis, providing a significant benefit for locally advanced high-risk prostate cancer patients
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