Dithiiranylmethyloxy azaxanthone shows potent anti-tumor activity via suppression of HER2 expression and HER2-mediated signals in HER2-overexpressing breast cancer cells

Nam, Jung Min; Jeon, Kyung Hwa; Kwon, Han Sung; Lee, Eun-Young; Jun, Kyu Yeon; Jin, Yeung Bae; Lee, Yun Sil; Na, Younghwa; Kwon, Youngjoo

doi:10.1016/j.ejps.2013.06.014

Cited by 14 publications

(8 citation statements)

References 60 publications

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“…Prior to assessing exosome uptake, the level of surface Her2 expressed on each cell line was analysed by quantitative flow cytometry. Our analysis of HeLa (cervical), BT20 (breast), HCC1954 (breast), SK-BR-3 (breast) cell lines confirmed the previously reported Her2-expression status of these cells; [31][32][33][34] HCC1954 and SK-BR-3 exhibit a high number of Her2 receptors per cell (3.6 × 10 6 and 4.2 × 10 6 , respectively) and HeLa and BT-20 only display 1-2 × 10 5 receptors per cell, which is comparable to expression in HEK293 at just under 1 × 10 5 receptors per cell (Fig. 3A).…”

Section: Targeting Of Anti-her2-scfv-exosomes To Tumour Cell Linessupporting

confidence: 89%

High affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles

Longatti¹,

Schindler²,

Collinson³

et al. 2018

Nanoscale

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Exosomes are extracellular vesicles that mediate cell-to-cell communication by transferring biological cargo, such as DNA, RNA and proteins. Through genetic engineering of exosome-producing cells or manipulation of purified exosomes, it is possible to load exosomes with therapeutic molecules and target them to specific cells via the display of targeting moieties on their surface. This provides an opportunity to exploit a naturally-occurring biological process for therapeutic purposes. In this study, we explored the potential of single chain variable fragments (scFv) as targeting domains to achieve delivery of exosomes to cells expressing a cognate antigen. We generated exosomes targeting the Her2 receptor and, by varying the affinity of the scFvs and the Her2 expression level on recipient cells, we determined that both a high-affinity anti-Her2-scFv (K≤ 1 nM) and cells expressing a high level (≥10 copies per cell) of Her2 were optimally required to enable selective uptake. We also demonstrate that targeting exosomes to cells via a specific cell surface receptor can alter their intracellular trafficking route, providing opportunities to influence the efficiency of delivery and fate of intracellular cargo. These experiments provide solid data to support the wider application of exosomes displaying antibody fragments as vehicles for the targeted delivery of therapeutic molecules.

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Section: Targeting Of Anti-her2-scfv-exosomes To Tumour Cell Linessupporting

confidence: 89%

High affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles

Longatti¹,

Schindler²,

Collinson³

et al. 2018

Nanoscale

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“…Previous reports have shown that ESE-1 regulates HER2 expression and that small molecule interference of the ESE-1/Sur2 interaction inhibits HER2-mediated downstream signaling [ 20 – 22 ]. Thus, we tested if the key mechanism by which ESE-1 mediates transformation is through regulation of HER2 protein expression and HER2 downstream signaling molecules.…”

Section: Resultsmentioning

confidence: 99%

“…Since HER2 signaling induces ESE-1 gene expression, and ESE-1 protein functions as a trans-activator of the HER2 promoter, it has been reported that HER2 and ESE-1 operate in a feed-forward mechanism [ 20 , 21 ]. It has also been shown that the anti-tumor effect of targeting the ESE-1/Sur2 interface is mediated through downregulation of HER2 and HER2 signaling in SKBR3 cells harvested 72 hours from the time of ESE-1/Sur2 disruption [ 22 ]. Here we have shown through transient and stable knockdown of ESE-1 protein that although the ESE-1/HER2 relationship exists, it is more complicated than previously appreciated and manifests distinct temporal responsiveness in a cell type-specific manner in the luminal B BT474 and the HER2 subtype SKBR3 cells.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, a positive feedback loop appears to exist between the HER2 proto-oncogene and ESE-1 , whereby HER2 signaling induces ESE-1 gene expression, and nuclear ESE-1 trans-activates the HER2 promoter [ 20 , 21 ]. In HER2 + SKBR3 breast cancer cells, disruption of ESE-1/Sur2 interaction with pharmacological inhibitors attenuates HER2-dependent signaling, at 72 hours [ 22 ]. But given the fact that Sur2 is a mediator protein commonly employed by the Pol II transcriptional machinery and that the small molecule inhibitor caused apoptosis (which is not observed with ESE-1 knockdown in transformed cell lines), the specific role of ESE-1 in the transformative process was not clear.…”

Section: Introductionmentioning

confidence: 99%

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ESE-1/ELF3 mRNA expression associates with poor survival outcomes in HER2+ breast cancer patients and is critical for tumorigenesis in HER2+ breast cancer cells

Kar

Gutierrez‐Hartmann

2017

Oncotarget

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ESE-1/Elf3 and HER2 appear to establish a positive feedback regulatory loop, but the precise role of ESE-1 in HER2+ breast tumorigenesis remains unknown. Analyzing public repositories, we found that luminal B and HER2 subtype patients with high ESE-1 mRNA levels displayed worse relapse free survival. We stably knocked down ESE-1 in HER2+ luminal B BT474 cells and HER2 subtype SKBR3 cells, which resulted in decreased cell proliferation, colony formation, and anchorage-independent growth in vitro. Stable ESE-1 knockdown inhibited HER2-dependent signaling in BT474 cells and inhibited mTOR activation in SKBR3 cells, but reduced Akt signaling in both cell types. Expression of a constitutively-active Myr-Akt partially rescued the anti-proliferative effect of ESE-1 knockdown in both cell lines. Furthermore, ESE-1 knockdown inhibited cyclin D1, resulting in a G1 delay in both cell lines. Finally, ESE-1 knockdown completely inhibited BT474 cell xenograft tumors in NOD/SCID female mice, which correlated with reduced in vitro tumorsphere formation. Taken together, these results reveal the ESE-1 controls transformation via distinct upstream signaling mechanisms in SKBR3 and BT474 cells, which ultimately impinge on Akt and cyclin D1 in both cell types to regulate cell proliferation. Particularly significant is that ESE-1 controls tumorigenesis and is associated with worse clinical outcomes in HER2 breast cancer.

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“…This blocking proved the high specificity of our new construct for the HER2 receptor. More proof of the high specificity of the cyclic dimer is given in Figure 7 B. MCF-7 cells present a low basal level of HER2 expression [ 41 ] and, upon treatment with the labeled cyclic Z HER2:342 dimer in the same conditions as the SKOV-3 cells, did not show any significant binding to the construct. The insignificant binding on MCF-7 cells indicates that in the absence of HER2 surface receptor, the cyclic dimer does not have any binding sites.…”

Section: Resultsmentioning

confidence: 99%

Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

et al. 2021

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Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific ZHER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic ZHER3:342-dimer with an apparent melting temperature, Tm, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (Kd ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (Kd ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.

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Dithiiranylmethyloxy azaxanthone shows potent anti-tumor activity via suppression of HER2 expression and HER2-mediated signals in HER2-overexpressing breast cancer cells

Cited by 14 publications

References 60 publications

High affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles

High affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles

ESE-1/ELF3 mRNA expression associates with poor survival outcomes in HER2+ breast cancer patients and is critical for tumorigenesis in HER2+ breast cancer cells

Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

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