HER2 siRNA Research Articles

Inhibition of Breast Cancer Cell Growth and Invasiveness by Dual Silencing of HER-2 and VEGF

Overexpression of HER-2 accounts for approximately 25% of all breast cancer cases, while 87.7% of HER-2 positive breast cancers are associated with upregulated VEGF. The objective of this study is to explore the combination therapy of blocking HER-2 and VEGF expressions simultaneously using siRNA. This is the first report to examine the effect of dual silencing of HER-2 and VEGF genes on tumor growth and invasiveness. We have designed nine HER-2 siRNAs and ten VEGF siRNAs, and identified potent siRNA which can silence the target gene up to 75-83.5%. The most potent HER-2 and VEGF siRNAs were used to conduct functional studies in HER-2 positive breast cancer cells. Tumor invasiveness properties including cell morphology change, in vitro migration, cell spreading, and adhesion to ECM were evaluated. In addition, cell proliferation and apoptosis were examined after the siRNA treatment. Our data demonstrated for the first time that HER-2 siRNA could inhibit cell migration and invasion abilities. Combination of HER-2 and VEGF siRNAs exhibited synergistic silencing effect on VEGF. Both HER-2 siRNA and VEGF siRNA showed significant inhibition on cell migration and proliferation. HER-2 siRNA also demonstrated dramatic suppression on cell spreading and adhesion to ECM, as well as induction of apoptosis. Dual silencing of HER-2 and VEGF exhibited significant cell morphology change, and substantial suppression on migration, spreading, cell adhesion, and proliferation. Our observations suggested that HER-2 positive breast cancer may be more effectively treated by dual inhibition of HER-2 and VEGF gene expressions using siRNA.

Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by synthetic small interfering RNA (siRNA), and is utilized in a wide range of fields including cancer gene therapy by down-regulating a specific target protein. In this study, for tumor-targeted siRNA delivery, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated tumor gene therapy in human nasopharyngeal KB cells, which overexpressed folate receptor (FR). NP-F was composed of cholesteryl-3β-carboxyamidoethylene- N-hydroxyethylamine (OH-Chol), Tween 80 and folate-poly(ethylene glycol)-distearoylphosphatidylethanolamine conjugate (f-PEG 2000-DSPE), and NP-P was substituted f-PEG 2000-DSPE in NP-F PEG 2000-DSPE for a non-targeting nanoparticle. The NP-F and siRNA complex (nanoplex) formed at a charge ratio (+/−) of 2/1 in the presence of 5 mM NaCl was injectable size and increased transfection efficiency in the cells. NP-F showed a significantly higher intracellular amount of siRNA and stronger localization of siRNA in the cytoplasm than NP-P. When Her-2 siRNA was transfected into cells by NP-F and NP-P, NP-F significantly inhibited tumor growth, and selectively suppressed Her-2 protein expression more than NP-P. In in vivo gene therapy, a NP-F nanoplex of Her-2 siRNA by intratumoral injection significantly inhibited tumor growth of KB xenografts compared with control siRNA, but a NP-P nanoplex did not. These results of the experiments have provided optimal conditions to form folate-linked nanoparticle complexes with siRNA for folate-targeted gene therapy.

Activity of lapatinib is independent of EGFR expression level in HER2-overexpressing breast cancer cells

Epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2/neu), members of the ErbB receptor tyrosine kinase family, are frequently overexpressed in breast cancer and are known to drive tumor growth and progression, making them promising targets for cancer therapy. Lapatinib is a selective competitive inhibitor of both the HER2 and EGFR tyrosine kinases. Although lapatinib showed significant activity in patients with HER2-positive breast cancer, the role of EGFR in the response of breast cancer to lapatinib has not been defined. Here, we examined the role of EGFR expression levels in the sensitivity of HER2-overexpressing breast cancer cells to lapatinib. Depletion of EGFR by EGFR small-interfering RNA knockdown did not affect lapatinib sensitivity in these cells, whereas treated HER2 siRNA knockdown cells became more resistant to lapatinib. We conclude that the in vitro activity of lapatinib is not dependent on EGFR expression level in HER2-overexpressing breast cancer cells.

Influence of human epidermal growth factor receptor-2 siRNA on chemosensitivity to cisplatin of human ovarian carcinoma cells: an in vitro experiment

To investigate the effects of RNA interference (RNAi) targeting human epidermal growth factor receptor 2 (HER-2) gene on the change of chemosensitivity to cisplatin of ovarian carcinoma cells. Methods Three kinds of HER-2 gene targeting siRNA, HER-2 siRNA I-III, were synthesized and the best one (HER-2 siRNA III) was screened. Human ovarian carcinoma cells of the line SKOV3 were cultured randomly divided into 3 groups: HER-2 siRNA III group, transfected with HER-2 siRNA III, non-specific siRNA group, transfected with non-specific siRNA III, and control group, without transfection. Cisplatin of the concentrations of 0, 0.05, 0.2. 0.4, 0.8, 1.0, 2.0, 10, and 20 microg/ml was added into the culture fluid for 24 h. MTT method was used to detect the proliferation rate of the SKOV3 cells. Other SKOV3 cells were divided into 3 groups: siRNA group, transfected with HER-2 siRNA III, cisplatin group, exposed to cisplatin, and HER-2 siRNA III and exposed to cisplatin. Annexin V method and flow cytometry were used to detect the apoptosis of the SKOV3 cells. The HER-2 gene expression was assessed by Western blotting. The chemosensitivity of transfected cells to cisplatin was measured by MTT. Western blotting was used to detect the protein expression of apoptosis related proteins: Bcl-2, surviving, XIAP, and Smac. After exposed to cisplatin, the cell survival rate decreased as the dose of cisplatin increased. The proliferation rate of the SKOV3 cells transfected with HER-2 siRNA III and exposed to 1 microg/ml cisplatin was (58 +/- 5)%, significantly lower than those of the nonspecific siRNA III transfection group [(65 +/- 6)%] and the control group [(68 +/- 3)%, both P < 0.01]. No significant difference in the cell survival rate was found between the control and nonspecific groups (P > 0.05). The apoptosis rates at different time point of the HER-2 siRNA III + cisplatin group were all higher than those of the other 2 groups (all P < 0.01). The protein expression levels of the antiapoptotic proteins Bcl-2, survivin, and XIAP were of the HER-2 siRNA III + cisplatin group were significantly lower than those of the cisplatin group, and the protein expression level of the apoptotic protein Smac of the HER-2 siRNA III + cisplatin group significantly higher than that of the cisplatin group (all P < 0.05). HER-2 siRNA III induces cell apoptosis significantly and increases the sensitivity of the human ovarian carcinoma cells to cisplatin. The mechanism of induction of cell apoptosis by HER-2 siRNA III + cisplatin may be related to the downregulation of Bcl-2, survivin and XIAP protein and the up-regulation of Smac protein.

QS287. HER2 Regulates Expression of Low Molecular Weight Cyclin E Isoforms in Breast Cancer

Objective: HER2 amplification and cyclin E overexpression have each been shown to be important prognostic indicators in breast cancer. In addition to overexpressing the full length (FL) cyclin E protein, some breast cancer cell lines and human breast cancers express up to 5 low molecular weight (LMW) isoforms which are generated by proteolytic processing of FL cyclin E by the serine protease elastase. These LMW forms have been demonstrated to be more tumorigenic than the FL protein. We hypothesized that HER2 overexpression may modulate cyclin E regulation in breast cancer, including generation of the LMW forms. Methods: HER2 expression was downregulated in the HER2-overepressing breast cancer cell lines MCF-7-HER-18 (HER18), SKBr3 and BT474 using 2 different mechanisms; treatment with trastuzumab dosed at 10 and 20 μg/ml or transfection with HER2 siRNA. The low-HER2 expressing cell line MCF-7 was used as a negative control. The effect on cyclin E expression was investigated using confocal immunofluorescence microscopy and western blot analysis. The effect of HER2 downregulation on cyclin E transcription was assessed using quantitative RT-PCR (qRT-PCR). To determine if there may be a clinically relevant relationship between these 2 proteins, HER2 and LMW cyclin E levels were assessed in 25 patient samples collected as part of an ongoing prospective protocol. Results: Confocal immunofluorescence microscopy demonstrated that HER2 downregulation using siRNA or inactivation by treatment with trastuzumab resulted in decreased total cyclin E expression. Because cyclin E is tightly regulated at the level of transcription, qRT-PCR was performed to determine the effects of HER2 downregulation on cyclin E mRNA synthesis. There was no difference (p=.18) in cyclin E mRNA synthesis for cells transfected with HER2 siRNA versus those transfected with random sequence and mock transfected controls suggesting that HER2 affects post-transcriptional processing of cyclin E. Having shown that the effect of HER2 on cyclin E expression is not at the level of transcription, western blot analysis was used to investigate the effects of HER2 downregulation on the LMW forms. Western blot demonstrated that the decrease in cyclin E was primarily in the LMW forms (figure) confirming the effect of HER2 to be post-transcriptional. Western blot analysis was then performed on lysates obtained from 25 patient samples. Nine (35%) patients had HER2-overexpressing tumors. Densitometry was used to quantify FL and LMW cyclin E expression. There was no significant difference in expression of FL cyclin E in HER2+ versus HER2- tumors (p=.87). In contrast, there was significantly higher expression of LMW cyclin E in HER2+ tumors (p=.04). Conclusions: HER2 downregulation affects post-transcriptional processing of cyclin E resulting in decreased expression of the more tumorigenic LMW forms. LMW forms of cyclin E are overexpressed in a subset of patients with HER2-overexpressing tumors. Further study into the mechanism by which HER2 effects post-transcriptional processing of FL cyclin E to its LMW isoforms may identify a role for LMW cyclin E as a second therapeutic target in patients with HER2 and LMW cyclin E-overexpressing breast cancer.

Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference

Gene silencing using short interfering RNA (siRNA) is fast becoming an attractive approach to probe gene function in mammalian cells. Although there have been some success in the delivery of siRNA using various methods, tracking their delivery and monitoring their transfection efficiency prove to be hard without a suitable tracking agent. Therefore, a challenge lies with the design of an efficient and at the same time, self-tracking, transfection agent for RNA interference. In this paper, chitosan nanoparticles (NPs) with encapsulated quantum dots (QDs) were synthesized and used to deliver HER2/neu siRNA. Using such a construct, the delivery and transfection of the siRNA can be monitored by the presence of fluorescent QDs in the chitosan NPs. Targeted delivery of HER2 siRNA to HER2-overexpressing SKBR3 breast cancer cells was shown to be specific with chitosan/QD NP surface labeled with HER2 antibody targeting the HER2 receptors on SKBR3 cells. Gene-silencing effects of the conjugated siRNA was also established using the luciferase and HER2 ELISA assays. These self-tracking siRNA delivery NPs will also aid in the monitoring of future gene silencing studies in vivo.

Specific inhibition of HER-2 expression in ovarian carcinoma cells by siRNA targeting HER-2

To investigate the effects of RNA interference (RNAi) targeting HER-2 gene on the biological traits of human ovarian carcinoma. siRNA specific to HER-2 gene was synthesized according to the sequence in the GenBank. Human ovarian carcinoma cells of the line SKOV-3 were cultured and divided into 3 groups: control group; non-specific group, transfected with non-specific siRNA; and specific group, transfected with specific HER-2 siRNA. On the 5th day after transfection cisplatin was added into the culture fluid. The expression of HER-2 mRNA and the expression of protein both before and after transfection were detected by RT-PCR and Western blotting. The cell apoptosis was assessed by flow cytometry. The chemosensitivity of transfected cells to cisplatin was determined by MTT method. Since the 3rd day after transfection the expression of HER-2 mRNA of the HER-2 siRNA group was suppressed till 10 days later. On the 7th day after transfection the expression rate of HER-2 protein of the HER-2 siRNA group was (25.5 +/- 0.8)%, significantly lower than those of the nonspecific siRNA group and control group, (95.7 +/- 0.8)% and (96.6 +/- 1.2)% respectively (both P < 0.001). On the 9th day after transfection no expression of HER-2 protein was found in the HER-2 siRNA group. The apoptosis rate of SKOV-3 cells increased time-dependently after transfection in the HER-2 siRNA group and reached the peak, (53.2 +/- 1.0)% on the 6th day, significantly higher than those of the non-specific siRNA group and control group, (4.1 +/- 0.3)% and (4.1 +/- 0.3)% respectively (both P < 0.001). After exposure to cisplatin for 24 hours, the survival rate of the HER-2 siRNA group was (58.4 +/- 0.8)%, significantly higher than those of the nonspecific siRNA group and control group, (68.0 +/- 0.6)% and (67.0 +/- 0.3)% respectively (both P < 0.001). siRNA targeting HER-2 synthesized in vitro and transfected into human ovarian carcinoma cells effectively suppresses the HER-2 expression, induces cell apoptosis, and increases the sensitivity to cisplatin of the cells. The successful application of HER-2 siRNA extends the list of available therapeutic modalities in the treatment of human ovarian cancer.