Anti-RhoA and Anti-RhoC siRNAs Inhibit the Proliferation and Invasiveness of MDA-MB-231 Breast Cancer Cells in Vitro and in Vivo

Abstract

Overexpression of RhoA or RhoC in breast cancer indicates a poor prognosis, due to increased tumor cell proliferation and invasion and tumor-dependent angiogenesis. Until now, the strategy of blockage of the Rho-signaling pathway has used either GGTI or HMG-CoA reductase inhibitors, but they are not specific to RhoA or RhoC inhibition. In this study, a new approach with anti-RhoA and anti-RhoC siRNAs was used to inhibit specifically RhoA or RhoC synthesis. Two transfections of either RhoA or RhoC siRNA (8.5 nM) into MDA-MB-231 human breast cancer cells or HMEC-1 endothelial cells induced extensive degradation of the target mRNA and led to a dramatic decrease in synthesis of the corresponding protein. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of Rho cell signaling. Finally, in a nude mouse model, intratumoral injections of anti-RhoA siRNA (100 microl at 85 nM) every 3 days for 20 days almost totally inhibited the growth and angiogenesis of xenografted MDA-MB-231 tumors. One may infer from these observations that specific inhibition of the Rho-signaling pathway with siRNAs represents a promising approach for the treatment of aggressive breast cancers.

A Novel Protein Geranylgeranyltransferase-I Inhibitor with High Potency, Selectivity, and Cellular Activity

“…The important role for geranylgeranylated proteins such as Rho in numerous diseases is increasingly being appreciated (36,41,42). In addition to potentially suppressing alternate prenylation of K-Ras, the impact of GGTI-DU40 on proteins such as RhoA/C and Cdc42 may prove to be beneficial in limiting oncogenesis by inhibiting proliferation and invasion (36,43,44).…”

“…In addition to potentially suppressing alternate prenylation of K-Ras, the impact of GGTI-DU40 on proteins such as RhoA/C and Cdc42 may prove to be beneficial in limiting oncogenesis by inhibiting proliferation and invasion (36,43,44). Rho activity has a clear function in angiogenesis (45), which in turn plays a critical role in the pathogenesis of tumors, rheumatoid arthritis, atherosclerosis, psoriasis, and diabetic retinopathy (46).…”

Histone H3 lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodeling

“…Tubule formation assay was performed as described previously (37). Briefly, HMEC-1 cells were transfected with siRNA for 24 h, and 4 × 10 4 trypsin-dissociated cells were resuspended in 0.5 mL fresh full medium and seeded into Matrigel-coated 24-well plates.…”

The Gα12/13 Family of Heterotrimeric G Proteins and the Small GTPase RhoA Link the Kaposi Sarcoma-associated Herpes Virus G Protein-coupled Receptor to Heme Oxygenase-1 Expression and Tumorigenesis

“…Plasmids carrying shRNAs sequences were constructed by inserting double-stranded shRNA oligonucleotides in the pSilencer 1.0-U6 plasmid following the manufacturer's instructions (Ambion). The plasmids pSshRNARho1 and pSshRNARho2 carrying shRNA sequences for RhoA were engineered by annealing the following single strand oligonucleotides: 5Ј-GACATGCTTGCTCATAGT-CTTCAAGAGAGACTATGAGCAAGCATGTCTTTTTT and 3Ј-AATTAAAAAAGACATGCTTGCTCATAGTCTCTCTT-GAAGACTATGAGCAAGCATGTCGGCC for pSshRNARho1 (41) and 5Ј-GAAACTGGTGATTGTTGGTTTCAAGAGAAC-CAACAATCACCAGTTTCTTTTTT and 3Ј-AATTAAAAAA-GAAACTGGTGATTGTTGGTTCTCTTGAAACCAACAAT-CACCAGTTTCGGCC for pSshRNARho2. pSshRNAG␣ 13.1 and pSshRNAG␣ 13.2 were constructed using the following sequences: shRNAG␣ 13.1 , 5Ј-GTCCAAGGAGATCGACAAATTCAAGA-GATTTGTCGATCTCCTTGGACTTTTTT and 3Ј-AATTAA-AAAAGTCCAAGGAGATCGACAAATCTCTTGAATTTGT-CGATCTCCTTGGACGGCC; shRNAG␣ 13.2 , 5Ј-GCAACGTG-ATCAAAGGTATTTCAAGAGAATACCTTTGATCACGTT-GCTTTTTT and 3Ј-AATTAAAAAAGCAACGTGATCAA-AGGTATTCTCTTGAAATACCTTTGATCACGTTGCG-GCC.…”