Silencing Of HER2 Research Articles

Aptamer-siRNA chimeras: Promising tools for targeting HER2 signaling in cancer.

RNA interference is a transformative approach and has great potential in the development of novel and more efficient cancer therapeutics. Immense prospects exist in the silencing of HER2 and its downstream genes which are overexpressed in many cancers, through exogenously delivered siRNA. However, there is still a long way to exploit the full potential and versatility of siRNA therapeutics due to the challenges associated with the stability and delivery of siRNA targeted to specific sites. Aptamers offer several advantages as a vehicle for siRNA delivery, over other carriers such as antibodies. In this review, we discuss the progress made in the development and applications of aptamer-siRNA chimeras in HER2 targeting and gene silencing. A schematic workflow is also provided which will provide ample insight for all those researchers who are new to this field. Also, we think that a mechanistic understanding of the HER2 signaling pathway is crucial in designing extensive investigations aimed at the silencing of a wider array of genes. This review is expected to stimulate more research on aptamer-siRNA chimeras targeted against HER2 which might arm us with potential effective therapeutic interventions for the management of cancer.

Activation of MAPK signalling results in resistance to saracatinib (AZD0530) in ovarian cancer.

SRC tyrosine kinase is frequently overexpressed and activated in late-stage, poor prognosis ovarian tumours, and preclinical studies have supported the use of targeted SRC inhibitors in the treatment of this disease. The SAPPROC trial investigated the addition of the SRC inhibitor saracatinib (AZD0530) to weekly paclitaxel for the treatment of platinum resistant ovarian cancer; however, this drug combination did not provide any benefit to progression free survival (PFS) of women with platinum resistant disease. In this study we aimed to identify mechanisms of resistance to SRC inhibitors in ovarian cancer cells. Using two complementary strategies; a targeted tumour suppressor gene siRNA screen, and a phospho-receptor tyrosine kinase array, we demonstrate that activation of MAPK signalling, via a reduction in NF1 (neurofibromin) expression or overexpression of HER2 and the insulin receptor, can drive resistance to AZD0530. Knockdown of NF1 in two ovarian cancer cell lines resulted in resistance to AZD0530, and was accompanied with activated MEK and ERK signalling. We also show that silencing of HER2 and the insulin receptor can partially resensitize AZD0530 resistant cells, which was associated with decreased phosphorylation of MEK and ERK. Furthermore, we demonstrate a synergistic effect of combining SRC and MEK inhibitors in both AZD0530 sensitive and resistant cells, and that MEK inhibition is sufficient to completely resensitize AZD0530 resistant cells. This work provides a preclinical rationale for the combination of SRC and MEK inhibitors in the treatment of ovarian cancer, and also highlights the need for biomarker driven patient selection for clinical trials.

Abstract 1530: RUNX1 positively regulates ErbB2/HER2 signaling pathway through modulating the expression of SOS1 in gastric cancer cells

Abstract Although runt-related transcription factor 1 (RUNX1) is well known for its context-dependent oncogenic properties in various malignancies, its role in gastric cancers has been poorly defined. Up-regulation of receptor tyrosine kinase (RTK) ErbB2/HER2 signaling pathway is encountered in the vast majority of gastric cancer cases and contributes to the initiation and maintenance of these cancer cells. This signaling cascade is partly yet arbitrary mediated by son of sevenless gene 1 (SOS1), which functions as an adaptor protein in RTK cascades. Herein we report that RUNX1 regulates ErbB2/HER2 signaling pathway in gastric cancer cells through transcriptionally regulating SOS1 expressions, rendering itself an ideal target in anticancer strategies. Mechanistically, RUNX1 interacts with the consensus RUNX1 binding sequence (5’-TGTGGT-3’) located in the proximal promoter region of SOS1 and positively regulates it. Short hairpin RNA (shRNA)-mediated knockdown of RUNX1 in the gastric cancer cell line MKN45 led to the decreased expression of SOS1 and of phosphorylated form of ErbB2/HER2 as well as the deactivation of its downstream targets such as AKT and ERK. RUNX1 knockdown subsequently induced cell cycle arrest at G0/G1 phase and successive apoptotic cell death in MKN45 cells. Silencing of HER2 or SOS1 in MKN45 cells unalterably suppressed the proliferation of these cancer cells, highlighting the importance of this ErbB2/HER2 signaling cascade in the maintenance of gastric cancer cells. We also found that SOS1 is one of the most consistently up-regulated genes in RUNX1-high expressing primary gastric cancer cells derived from previously reported human clinical samples. These data collectively indicates that inhibition of RUNX1 could be a legitimate therapeutic choice in the management of gastric cancers. Lastly, we examined the efficacy of a novel small molecule specifically binds and inhibits RUNX1 (we named it as CM). Intriguingly, CM was exceptionally effective against MKN45 cells (IC50 value at 403.5 nM). Besides, CM was well-tolerated in mice and fabulously suppressed the growth of xenotransplanted MKN45 cells in immunodeficient mice in vivo. Taken together, our work identified a novel interaction of RUNX1 and ErbB2/HER2 signaling pathway, paving a new way for the management of dismal-prognostic advanced stage HER2-positive gastric cancer patients. Citation Format: Yoshihide Mitsuda, Ken Morita, Shintaro Maeda, Kensho Suzuki, Gengo Kashiwazaki, Junichi Taniguchi, Toshikazu Bando, Hiroshi Sugiyama, Souichi Adachi, Yasuhiko Kamikubo. RUNX1 positively regulates ErbB2/HER2 signaling pathway through modulating the expression of SOS1 in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1530. doi:10.1158/1538-7445.AM2017-1530

P0059 Induction of apoptosis in breast cancer cells through combined intracellular delivery of a tumour-suppressor gene and small interfering RNAs against growth factor receptor and anti-apoptotic genes

Background The mechanisms underlying the development of breast cancer are complex and vary among individual tumours. These mechanisms include genetic and epigenetic alterations and resulting changes in the activity of signalling pathways. Current treatment strategies for metastatic breast cancer (MBC) depend upon patient and tumour classification; menopausal status, hormone receptor status, and HER2 status can all be considered, as can site of metastatic disease (bone or soft tissue). The choice of treatment in metastatic disease usually involves systemic endocrine therapy or cytotoxic chemotherapy, with an anti-HER2 agent when appropriate. Novel agents are needed because many of the current therapies have limitations. These include drug resistance, lack of target receptor expression in tumours (e.g., only 25% of breast cancer tumours have HER2 expression), lack of specificity, and relatively small improvements in survival. Advances in the understanding of the aetiology and biology of breast cancer have identified key targets among the multiple signalling pathways involved in the development, growth, and survival of breast cancer cells. As such, targeted therapies are among the most promising new agents for the treatment of breast cancer. Methods The main objective of the project is the restoration of p53 activity and the silencing of HER2, EGFR, and IGF1 genes through the delivery of normal p53 gene and specific validated siRNA, respectively, using carbonate apatite nanoparticles both in vitro and in vivo. Concurrently, the sensitivity of breast cancer cells to the traditional anti-cancer drugs, such as doxorubicin, cisplatin, and paclitaxel will be tested following individually and combined delivery p53 gene and the siRNAs against both growth factor receptor genes and anti-apoptotic genes. Findings The in vitro analysis of siRNAs together and in combination with p53 and anticancer drugs helped to identify potential targets for the treatment of breast cancer. Interpretation Preclinical studies in animal models of breast cancer should be done through tumour-targeted delivery of siRNA in combination with passively diffusible anti-cancer drugs and p53.

The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells

About 20% of breast cancers are characterized by amplification and overexpression of the HER2 oncogene. Although significant progress has been achieved for treating such patients with HER2 inhibitor trastuzumab, more than half of the patients respond poorly or become resistant to the treatment. Since the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to trastuzumab resistance. We integrated aCGH data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes with several phenotypic endpoints in a panel of trastuzumab responding and non-responding HER2 positive breast cancer cells. Silencing of HER2 caused a greater growth arrest and apoptosis in the responding compared to the non-responding cell lines, indicating that the resistant cells are inherently less dependent on the HER2 pathway. Several other genes in the amplicon also showed a more pronounced effect when silenced; indicating that expression of HER2 co-amplified genes may be needed to sustain the growth of breast cancer cells. Importantly, co-silencing of STARD3, GRB7, PSMD3 and PERLD1 together with HER2 led to an additive inhibition of cell viability as well as induced apoptosis. These studies indicate that breast cancer cells may become addicted to the amplification of several genes that reside in the HER2 amplicon. The simultaneous targeting of these genes may increase the efficacy of the anti-HER2 therapies and possibly also counteract trastuzumab resistance. The observed additive effects seem to culminate to both apoptosis and cell proliferation pathways indicating that these pathways may be interesting targets for combinatorial treatment of HER2+ breast cancers.

Abstract A184: Integrative molecular and functional profiling of HER2 amplified breast cancers to identify new gene targets.

Abstract Up to 15% of all breast cancers are defined as HER2+ve where overexpression is due to amplification of the HER2 oncogene. Treatment of patients with these clinically aggressive tumors has been dramatically improved by the use of trastuzumab and lapatinib. However a significant proportion of patients' tumors remain resistant to these therapies. Therefore new biomarkers and additional therapeutic targets are urgently required for what remains a clinically challenging and biologically heterogeneous disease. We describe an integrated genetic, transcriptomic and functional analysis of a wide panel of genes amplified and overexpressed in HER2 amplified breast tumors to identify new gene targets. Molecular profiling of 45 sporadic primary HER2 amplified breast cancers, 14 commercially available HER2 amplified and 9 non HER2 amplified breast cancer cell lines was performed. Genome wide copy number and transcriptomic array data were integrated and identified 511 genes that were significantly overexpressed when amplified in the tumors. The functional significance of 369 of these genes, which were recurrently amplified in the cell lines, was examined using parallel high throughput siRNA screens. Multiple genetic dependencies were observed. We demonstrate that loss of cell viability following silencing of HER2 by multiple siRNAs significantly correlated with sensitivity to lapatinib and other HER2 targeted agents. We also show that silencing of TFAP2C caused significant loss of cell viability when amplified and overexpressed in multiple HER2 amplified breast cancer cell lines. Down regulation of HER2 expression and cell apoptosis was also observed. The potential clinical value of TFAP2C overexpression for patients with HER2+ve breast cancer is currently being explored in adjuvant HER2+ breast cancer trials of chemotherapy +/− HER2 targeted agents. We propose that TFAP2C amplification and overexpression represents a genetic dependency in HER2+ breast cancer and is a clinically relevant candidate biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A184.

Silencing of Her2, CCNB1 and pKC Genes by siRNA Results in Prolonged Retardation of Neuroblastoma Cell Division

Deregulation of the expression of the genes that are involved in the control of the cell cycle impairs cellular differentiation and leads to cell death. This process can result in uncontrollable cell proliferation and, subsequently, cancer development. In this study, we examined the effect of the silencing of cancer-related genes by small interfering RNAs (siRNA) targeted at mRNAof Her2, cyclin B1 (CCNB1), and protein kinase C(PKC) on the proliferation of human cancer cells of different origins. Maximum silencing ofCCNB1,Her2(in KB-3-1, SK-N-MC, MCF-7 cells), andPKC(in MCF-7 cells) was achieved 72 h after transfection of the corresponding siRNAs, and 12 days after the transfection, the initial levels of the target mRNAs were fully recovered. Silencing ofHer2,CCNB1,andPKCdifferently effected the proliferation of the cell lines under study. The most pronounced antiproliferative action of the investigated siRNAs was observed in neuroblastoma SK-N-MC cells (3 - 10-fold reduction in the proliferation rate) even after the recovery of the initial levels of expression ofthe Her2,CCNB1, andPKС genes. The obtained data indicate that theCCNB1 andPKCgenes can be used as targets in the development of drugs for neuroblastoma treatment.

RNAi‐mediated downregulation of uPAR synergizes with targeting of HER2 through the ERK pathway in breast cancer cells

Overexpression of urokinase plasminogen activator receptor (uPAR) or HER2 (erbB-2) in breast cancer is associated with a poor prognosis. We previously reported that gene amplification and overexpression of HER2 and uPAR occur in 70% of HER2-amplified tumor cells from blood or tissue of patients with breast cancer. In this study, we first examined whether depletion of HER2 and uPAR synergized in suppression of the growth of breast cancer cells that overexpress both HER2 and uPAR (SKBR3 and ZR 751). The results showed that depletion of either HER2 or uPAR by RNA interference suppressed cell growth and induced cell apoptosis, but that these effects were significantly enhanced in cells depleted of both HER2 and uPAR. Mechanistic analysis demonstrated that silencing of HER2 and uPAR caused suppression of MAPK signal pathways, resulting in decrease of ERK activity and prompting a high p38/ERK activity ratio. The level of the phosphorylated form of ERK was decreased in cells depleted of HER2, uPAR or both, and the effect in cells depleted of both is the most evident. Moreover, downregulation of uPAR synergized with trastuzumab to suppress the growth and induce apoptosis of SKBR3 and ZR 751 cells. uPAR RNAi significantly enhanced the effect of trastuzumab on inhibition of MAPK signal pathways. In conclusion, targeting HER2 and uPAR has a synergistic inhibitory effect on breast cancer cells. Our results provide evidence that simultaneous downregulation of HER2 and uPAR may offer an effective tool for breast cancer therapy.

Silencing of the CCNB1, Her2, and PKC genes by small interfering RNA differently retards the division of different human cancer cell lines

Deregulation of genes encoding proteins responsible for cell cycle control frequently accompanies cell malignization and switches the cell program from differentiation and apoptosis to uncontrollable proliferation. We used siRNAs targeted to HER2, protein kinase C (PKC), and cyclin B1 (CCNB1) mRNAs to evaluate the therapeutic potential of the suppression of genes coding for key cell cycle regulators in different human cancer cells. The CCNB1, HER2, or PKC mRNA levels were efficiently reduced within 48 h after transfection with siCycB1, siHER2 or siPKC, respectively. Silencing of HER2, PKC, and CCNB1 substantially reduced the growth rates of all cell lines under study except HL-60 but did not affect cell death or apoptosis. The most pronounced inhibition of cell division was induced by siCycB1 in SK-N-MC cells and by siPKC in MCF-7 cells. We conclude that the selected siRNAs inhibit tumor cell division, and the investigated genes can be promising targets in cancer treatment.