HER2-overexpressing Cancer Cells Research Articles

Biofunctional quantum dots as fluorescence probe for cell-specific targeting

We describe here the synthesis, characterization, bioconjugation, and application of water-soluble thioglycolic acid TGA-capped CdTe/CdS quantum dots (TGA-QDs) for targeted cellular imaging. Anti-human epidermal growth factor receptor 2 (HER2) antibodies were conjugated to TGA-QDs to target HER2-overexpressing cancer cells. TGA-QDs and TGA-QDs/anti-HER2 bioconjugates were characterized by fluorescence and UV–Vis spectroscopy, X-ray diffraction (XRD), hydrodynamic sizing, electron microscopy, and gel electrophoresis. TGA-QDs and TGA-QDs/anti-HER2 were incubated with cells to examine cytotoxicity, targeting efficiency, and cellular localization. The cytotoxicity of particles was measured using an MTT assay and the no observable adverse effect concentration (NOAEC), 50% inhibitory concentration (IC50), and total lethal concentration (TLC) were calculated. To evaluate localization and targeting efficiency of TGA-QDs with or without antibodies, fluorescence microscopy and flow cytometry were performed. Our results indicate that antibody-conjugated TGA-QDs are well-suited for targeted cellular imaging studies.

Structural Basis for Eliciting a Cytotoxic Effect in HER2-Overexpressing Cancer Cells via Binding to the Extracellular Domain of HER2

Human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase directly linked to the growth of malignancies from various origins and a validated target for monoclonal antibodies and kinase inhibitors. Utilizing a new approach with designed ankyrin repeat proteins (DARPins) as alternative binders, we show that binding of two DARPins connected by a short linker, one targeting extracellular subdomain I and the other subdomain IV, causes much stronger cytotoxic effects on the HER2-addicted breast cancer cell line BT474, surpassing the therapeutic antibody trastuzumab. We determined crystal structures of these DARPins in complex with the respective subdomains. Detailed models of the full-length receptor, constrained by its rigid domain structures and its membrane anchoring, explain how the bispecific DARPins connect two membrane-bound HER2 molecules, distorting them such that they cannot form signaling-competent dimers with any EGFR family member, preventing any kinase dimerization, and thus leading to a complete loss of signaling.

CLC604 preferentially inhibits the growth of HER2-overexpressing cancer cells and sensitizes these cells to the inhibitory effect of Taxol in vitro and in vivo

HER2 has become a solicitous therapeutic target in metastatic and clinical drug-resistant cancer. Here, we evaluated whether or not 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) and its furopyrazole and thienopyrazole analogues repress the expression of the HER2 protein. Among the test compounds, (1-benzyl-3-(p-hydroxymethylphenyl)-5-methylfuro[3,2-c]pyrazol) (CLC604), an isosteric analogue of YC-1, significantly suppressed the expression of HER2, and preferentially inhibited cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Our results revealed that CLC604 reduced HER2 expression through a post-transcriptional mechanism and involvement of proteasomal activity. CLC604 disrupted the association of 90-kDa heat shock protein (Hsp90) with HER2 resulting from the inhibition of Hsp90 ATPase activity. Moreover, we found that CLC604 significantly enhanced the antitumor efficacy of clinical drugs against HER2-overexpressing tumors and efficiently reduced HER2-induced drug resistance in vitro and in vivo. These findings suggest that CLC604 should be developed further as a novel antitumor drug candidate for the treatment of drug-resistant cancer.

Catecholamine-Induced β2-Adrenergic Receptor Activation Mediates Desensitization of Gastric Cancer Cells to Trastuzumab by Upregulating MUC4 Expression

Trastuzumab is currently used for patients with Her2(+) advanced gastric cancer. However, the response rate to trastuzumab among the patients is low. The molecular mechanisms underlying trastuzumab resistance in gastric cancer are unknown. Our in vitro data show that activation of β2-adrenergic receptor (β2-AR) triggered by catecholamine caused "targeting failure" of trastuzumab in gastric cancer cells. The antitumor activities of trastuzumab were significantly impeded by chronic catecholamine stimulation in gastric cancer cells and in the mice bearing human gastric cancer xenografts. Mechanistically, catecholamine induced upregulation of the MUC4 expression at both transcription and protein levels via activating STAT3 and ERK. The effects of catecholamine could be effectively blocked by β2-AR antagonist ICI-118,551, indicating that β2-AR-mediated signaling pathway plays a key role in upregulation of MUC4, which was previously demonstrated to interfere with the recognition and physical binding of trastuzumab to Her2 molecules. Moreover, a significant elevation of the MUC4 level was observed in the xenograft tissues in nude mice chronically treated with isoproterenol. Knockdown of MUC4 restored the binding activities of trastuzumab to Her2-overexpressing gastric cancer cells. In addition, coexpression of β2-AR and MUC4 were observed in gastric cancer tissues. Our data indicated a novel trastuzumab resistance mechanism, by which catecholamine-induced β2-AR activation mediates desensitization of gastric cancer cells to trastuzumab through upregulating the MUC4 expression.

Abstract LB-8: A novel remote loading method with solubility gradient to encapsulate effevtive amount of taxanes into liposomes.

Abstract Targeted liposomal drug delivery is considered to enhance the therapeutic effects of anti-cancer agents because high amount of drug could be administered with least side effects. However, water-insoluble hydrophobic and non-weak base chemical compounds such as paclitaxel (PTX) are not suitable for efficient encapsulating. We conferred water-solubility of PTX by coupling glucose at 7-OH to produce 7-alpha-glucosyloxyacetylpaxcitacel (gPTX). gPTX showed enhanced water-solubility with the 40 % ethylene glycol (EG) in PBS and with the CEP buffer (Cremophor EL and Ethanol in PBS). The maximal solubility of gPTX in these solvents was 1 and 20 mg/ml, respectively. We prepared gPTX encapsulated liposome (gPTX-L) with remote loading method by solubility gradient between 40 % EG and CEP buffer. gPTX efficiently encapsulated into liposomes. The encapsulation efficiency and loading efficiency were 72.9 % and 8.5×10−2 drug mol / initial lipid mol. Then, trastuzumab was conjugated on the surface of gPTX-L to prepare immunoliposome (gPTX-IL) for targeting HER2 overexpressing cancer cells. The particle size of gPTX-IL was under 150 nm and Z potential was -3.07 mV. We evaluated IC50 values by MTT assay after 72 hours exposure. The IC50 values of PTX, gPTX, gPTX-L and gPTX-IL for SK-BR-3 cells were 5.5, 18.9, 6.6 and 5.3 nM, respectively. gPTX-IL exhibited the highest toxicity in these paclitaxel formulations. Similarly, the IC50 values for HT-29 cells were 1.3, 11.0, 7.6 and 6.8 nM, respectively. We then evaluated the time required for each formula to inhibit the growth at the half maximal level (IT50) at the minimum concentration to show maximal cytotoxicity (IC100) after 72 hours exposure. gPTX-IL showed the shortest IT50 values of 1.6 and 1.1 h in SK-BR-3 and HT-29 cells respectively. We then evaluated in vivo distribution of liposome and immunoliposome encapsulating HSA conjugated Cy5.5 in ICR-nu/nu mice bearing HT-29 cell tumors. Both liposome and immunoliposome accumulated at the tumor site, but immunoliposome exhibited less accumulation at liver than liposome. Based on these results, we then evaluated the anti-cancer efficacy of the formula in vivo using tumor bearing ICR-nu/nu mice. While gPTX-L and free trastuzumab did not inhibit tumor growth, gPTX-IL decreased tumor volume and inhibited tumor growth with low side effects at the dose of 150 mg/kg gPTX, which was the lethal dose of naked gPTX exhibiting 25% survival rate of mice one week after the injection of gPTX. Collectively, the gPTX-encapsulated immunoliposome with novel encapsulation strategy should be an excellent candidate of highly efficient drug delivery system of cancer treatment targeting HER2. Citation Format: Tsukasa Shigehiro, Tomonari Kasai, Akifumi Mizutani, Hiroshi Murakami, Katsuhiko Mikuni, Tadakatsu Mandai, Hiroki Hamada, Masaharu Seno. A novel remote loading method with solubility gradient to encapsulate effevtive amount of taxanes into liposomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-8. doi:10.1158/1538-7445.AM2013-LB-8

Abstract 3271: Tumor priming with cyclophosphamide increases delivery and activity of Her2-targeted liposomal doxorubicin (MM-302) in preclinical models of breast cancer.

Abstract MM-302 is a HER2-targeted liposomal doxorubicin formulation designed to target doxorubicin to HER2-overexpressing cancer cells while limiting uptake into non-target cells. The combination of doxorubicin with cyclophosphamide has been widely used in both the early- and late-stage breast cancer settings. Recent clinical trials have shown promising response rates to the combination of cyclophosphamide, pegylated liposomal doxorubicin (PLD) and trastuzumab in HER2-positive disease. The purpose of this study is to investigate the effects of cyclophosphamide on the tumor delivery of MM-302, and to evaluate the anti-tumor activity of this drug combination in preclinical models of breast cancer. Methods: Biodistribution studies were carried out in BT474-M3, SUM190, and MDA-MB-453 tumor-bearing mice to assess the delivery of MM-302, PLD and free doxorubicin as single agents or in combination with cyclophosphamide at different dosing schedules. The total doxorubicin within tumors was quantified by HPLC and, microscopically, by automated image analysis in order to determine the extent of doxorubicin-positive nuclei. Automated image analysis was also used to quantify tumor cell apoptosis, changes in tumor cell density, and induction of DNA-damage in response to drug treatment. Activity studies in BT474-M3 tumor-bearing mice were performed to evaluate the ability of the different dosing regimens to inhibit tumor growth. Results: Pre-dosing of tumors with cyclophosphamide significantly increased the delivery of MM-302 to tumors. The increase in delivery was tumor-specific, and the cyclophosphamide pre-dose had no effect on doxorubicin accumulation to non-target tissues such as heart or skin. Pre-treatment of tumors with cyclophosphamide also increased the delivery of PLD to tumors, but not of free doxorubicin. However, the increase in nuclear doxorubicin delivery via MM-302 compared to PLD was significantly higher in the HER2-positive breast cancer models analyzed. Histological analysis of the cyclophosphamide-treated tumors suggests that induction of tumor cell apoptosis and reduction of overall tumor cell density are among the mechanisms for improved MM-302 delivery and tumor cell response to doxorubicin in this preclinical model. Finally, treatment of BT474-M3 tumors with cyclophosphamide followed by MM-302 resulted in a significantly higher tumor growth inhibition compared to both single agents as well as compared to a MM-302 and cyclophosphamide co-injection regimen. Conclusions: Rational combination of MM-302 with cyclophosphamide is an active and tolerable combination in preclinical models that enhances the HER2-targeting properties of MM-302. This study suggests that cyclophosphamide may be a promising combination partner for MM-302 in clinical studies. Citation Format: Elena Geretti, Shannon C. Leonard, Daniel F. Gaddy, Nancy Dumont, Thomas J. Wickham, Bart S. Hendriks. Tumor priming with cyclophosphamide increases delivery and activity of Her2-targeted liposomal doxorubicin (MM-302) in preclinical models of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3271. doi:10.1158/1538-7445.AM2013-3271

Mollugin inhibits proliferation and induces apoptosis by suppressing fatty acid synthase in HER2‐overexpressing cancer cells

Mollugin is a naphthohydroquine found in the roots of Rubia cordifolia, and has been reported to have a variety of biological activities, including anti-inflammatory and apoptotic effects. In the present study, we investigated the molecular mechanisms by which mollugin exerts anti-tumor effect in HER2-overexpressing cancer cells. Our results showed that mollugin exhibited potent inhibitory effects on cancer cell proliferation, especially in HER2-overexpressing SK-BR-3 human breast cancer cells and SK-OV-3 human ovarian cancer cells in a dose- and time-dependent manner without affecting immortalized normal mammary epithelial cell line MCF-10A. Furthermore, we found that a blockade of Akt/SREBP-1c signaling through mollugin treatment significantly reduced FAS expression and subsequently suppressed cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Mollugin treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level, potentially in part through suppression of NF-κB activation. The combination of mollugin with a MEK1/2 inhibitor may be required in order to achieve optimal efficacy in HER2-overexpressing cancers. These data provide evidence that mollugin inhibits proliferation and induces apoptosis in HER2-overexpressing cancer cells by blocking expression of the FAS gene through modulation of a HER2/Akt/SREBP-1c signaling pathway. Our findings suggest that mollugin is a novel modulator of the HER2 pathway in HER2-overexpressing cancer cells with a potential role in the treatment and prevention of human breast and ovarian cancer with HER2 overexpression.

Multifunctional graphene quantum dots-conjugated titanate nanoflowers for fluorescence-trackable targeted drug delivery

Multifunctional graphene quantum dots (GQDs) conjugated layered protonated titanate (LPT) nanoflowers have been developed as a promising system for fluorescence imaging and targeted drug delivery. The layered structure of the titanate nanoflowers provides a high specific area for loading drugs. The negatively charged nanocarrier shows a high loading capacity for doxorubicin (DOX). The fluorescence of GQDs reveals the intracellular localization of nanocarriers, suggesting that the uptake is via active endocytosis. Anti-HER2 labelling not only enables rapid uptake into HER2-overexpressing cancer cells, but also improves the nuclear accumulation of DOX. While the drug-free nanocarriers are highly biocompatible for up to 200 μg mL−1, the DOX loaded nanocarriers are more potent than free DOX in anticancer therapy. It is demonstrated that the anti-HER2–GQD–LPT system is a promising platform for simultaneous cancer imaging and anticancer therapeutics.

Trastuzumab-Doxorubicin Conjugate Provides Enhanced Anti-Cancer Potency and Reduced Cardiotoxicity

Since trastuzumab monotherapy for treatment of breast cancer with HER2/ErbB2 over-expression has been shown to have limited efficacy, combined treatment of trastuzumab with chemotherapy is widely practiced in clinic. However, certain combination treatments of trastuzumab and chemotherapy (i.e. doxorubicin) are not recommended due to high risk of cardiotoxicity. Antibody-drug conjugates (ADCs) offer selective delivery of cytotoxic agents into targeted cancer cells, thereby allowing for reduced general cellular cytotoxicity caused by chemotherapeutic agents through antibody mediated specific recognition of tumor antigens. In this study, we constructed a trastuzumab-doxorubicin conjugate (T-Dox) using a thioether linkage and characterized both biophysical stability and anti-cancer potency of the T-Dox using a panel of HER2 expressing cancer cell lines. The T-Dox conjugate showed significantly improved anti-cancer potency in comparison with trastuzumab. The results demonstrated for the first time that there were significant differences in the uptake of T-Dox among high HER2 expression cancer cells and higher T-Dox uptake also showed stronger anti-cancer potency. Similar to trastuzumab, T-Dox selectively bound to HER2 overexpressing cancer cells and low HER2 expression cells had no detectable uptake of T-Dox. Consistent to the uptake data, human cardiomyocyte cells had no detectable HER2 expression and T-Dox showed minimal cytotoxic effects. On the contrary, a treatment with combination of trastuzumab and doxorubicin showed severe cytotoxicity to human cardiomyocytes (>90% cell death after 3 day exposure). This study demonstrated that trastuzumab conjugated with doxorubicin (T-Dox) can provide valuable alternative to the combination treatment with doxorubicin and trastuzumab for high HER2 expressing cancer patients.

Ganoderma tsugaeExtract Inhibits Growth of HER2-Overexpressing Cancer Cells via Modulation of HER2/PI3K/Akt Signaling Pathway

Ganoderma, also known as Lingzhi or Reishi, has been used for medicinal purposes in Asian countries for centuries. It is a medicinal fungus with a variety of biological properties including immunomodulatory and antitumor activities. In this study, we investigated the molecular mechanisms by which Ganoderma tsugae (GT), one of the most common species of Ganoderma, inhibits the proliferation of HER2-overexpressing cancer cells. Here, we show that a quality assured extract of GT (GTE) inhibited the growth of HER2-overexpressing cancer cells in vitro and in vivo and enhanced the growth-inhibitory effect of antitumor drugs (e.g., taxol and cisplatin) in these cells. We also demonstrate that GTE induced cell cycle arrest by interfering with the HER2/PI3K/Akt signaling pathway. Furthermore, GTE curtailed the expression of the HER2 protein by modulating the transcriptional activity of the HER2 gene and the stability/degradation of the HER2 protein. In conclusion, this study suggests that GTE may be a useful adjuvant therapeutic agent in the treatment of cancer cells that highly express HER2.

Bright far-red/near-infrared fluorescent conjugated polymer nanoparticles for targeted imaging of HER2-positive cancer cells

We report the design and synthesis of a far-red/near-infrared (FR/NIR) fluorescent conjugated polymer, PFDBT-POSS, bearing poly{[9,9-di(hexyl)fluorene]-alt-co-[4,7-bis(thiophen-2-yl)-2,1,3-benzothiazole]} (PFDBT) as the backbone and bulky polyhedral oligomeric silsesquioxane (POSS) as side chains. PFDBT-POSS based nanoparticles (NPs) with maleimide (Mal) groups on the surface are prepared by encapsulating the polymer using DSPE-PEG2000 and DSPE-PEG2000-Mal as matrix. The obtained PFDBT-POSS-Mal NPs show a maximum absorption at ∼536 nm and a FR/NIR emission centred at ∼687 nm. It has a quantum yield (QY) of 13.6% in water, which is 6-fold higher than that of PFDBT-Mal NPs (QY = 2%), indicating that incorporation of POSS segments as pendants can efficiently suppress fluorescence quenching. The presence of Mal functional groups allows for efficient bioconjugation with anti-HER2 affibody by click reaction. The resultant PFDBT-POSS-Affibody serves as a reliable fluorescent probe with low cytotoxicity and high photostability in targeted detection and cellular imaging of HER2-overexpressed cancer cells using SKBR-3 as an example. Of particular interest is that the absorption maximum of PFDBT-POSS-Mal NPs overlaps well with the commercial 543 nm laser line, which ensures the most efficient utilization of the light harvesting property of the probe to generate bright fluorescence signals. This study provides an efficient strategy to design bright FR/NIR fluorescent CP NPs with long wavelength absorption and emission for biological applications.

HER2-Akt signaling in regulating COP9 signalsome subunit 6 and p53

HER2/neu oncogene is frequently overexpressed in various types of cancer, and the (PI3K)-Akt signaling pathway is often activated in HER2-overexpressing cancer cells. CSN6, subunit 6 of the COP9 signalosome complex, is pivotal in regulating MDM2 to destabilize p53, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to CSN6 regulation, and that Akt is a positive regulator of CSN6. Ectopic expression of Akt can increase the expression of CSN6; accordingly, Akt inhibition leads to CSN6 destabilization. Mechanistic studies show that Akt causes CSN6 phosphorylation at Ser 60, which, in turn, reduces ubiquitin-mediated protein degradation of CSN6. Significantly, Akt’s positive impact on CSN6 elevation translates into p53 degradation, potentiating transformational activity and increasing DNA damage. Akt inhibition can attenuate these defects caused by CSN6. These data suggest that Akt is an important positive regulator of CSN6, and that activation of Akt in many types of cancer could lead to abnormal elevation of CSN6 and result in downregulated p53 and increased DNA damage, which promotes cancer cell growth.

Suppression of FUT1 attenuates cell proliferation in the HER2-overexpressing cancer cell line NCI-N87

Lewis Y (LeY) antigen is an oligosaccharide that is highly expressed at the cell surface in various human cancers. Increased LeY expression activates epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and promotes cell proliferation in EGFR-overexpressing cells. However, the effect of downregulation of LeY expression on cell proliferation in HER2-overexpressing cells remains unknown. FUT1 encodes α1,2-fucosyltransferase, a key enzyme for LeY synthesis. We knocked down FUT1 by short interfering RNA (siRNA) in four HER2-overexpressing human cancer cell lines, including NCI-N87, MKN7, SKBr3 and BT474. We investigated whether downregulation of LeY and alteration in the glycosylation status of these cells affect cell proliferation and HER2 activation. Knocking down FUT1 expression markedly inhibited proliferation of NCI-N87, which highly expressed EGFR and was sensitive to EGFR deprivation. Furthermore, FUT1 siRNA downregulated the total amount of HER2 protein, phosphorylation of HER2 and EGFR, and phosphorylation of extracellular signal-regulated kinase (ERK) in this cell line. Moreover, the marked downregulation of phosphorylation of HER2 and ERK was observed following short-time EGF-stimulation. These effects were not observed in the other three cell lines. Our results suggest that knockdown of FUT1 downregulates HER2 signaling via EGFR downregulation. FUT1 may serve as a new molecular target for HER2-overexpressing human cancers with activated EGFR signaling.

The potential for trastuzumab emtansine in human epidermal growth factor receptor 2 positive metastatic breast cancer: latest evidence and ongoing studies.

The treatment of breast cancer that is driven by amplification and overexpression of human epidermal growth factor receptor 2 (HER2) has been drastically improved by the development of HER2-targeted therapies including trastuzumab and lapatinib. While outcomes for patients diagnosed with HER2-positive breast cancer have been greatly impacted by these therapies, treatment resistance is common and toxicity to standard regimens remains a therapeutic challenge. Trastuzumab emtansine (T-DM1) is a novel antibody drug conjugate that consists of the HER2-targeted monoclonal antibody, trastuzumab, joined via a stable linker to a derivative of maytansine, a highly potent cytotoxic chemotherapy. While other antibody drug conjugates have been developed clinically, this is the first in its class that maintains the antitumor properties of the HER2-targeted antibody, trastuzumab, and also avoids release of the chemotherapy until the molecule is taken up inside the HER2-overexpressing cancer cell. Several phase I studies have shown T-DM1 is safe, tolerable and has activity in trastuzumab- and lapatinib-pretreated breast cancer. Moreover, phase II studies are now being reported that confirm its safety and clinical efficacy in both the frontline and heavily pretreated settings. Preliminary data from phase II studies evaluating its use in combination with other cytotoxics have also been reported and several large phase III trials are underway to evaluate its use in the HER2-positive metastatic breast cancer setting. This paper aims to provide a detailed review of the preclinical and clinical evidence relating to the mechanism of action, efficacy and safety of T-DM1 for the treatment of HER2-positive breast cancer.

SV40 T/t-common polypeptide enhances the sensitivity of HER2-overexpressing human cancer cells to anticancer drugs cisplatin and doxorubicin

HER2-overexpressing cancer cells are resistant to cisplatin (CDDP) and doxorubicin (DXR). Here we report that SV40 T/t-common polypeptide could specifically sensitize HER2-overexpressing cancer cells to CDDP and DXR and specifically enhance CDDP- or DXR-induced apoptosis in these cells. This activity of T/t-common may be attributed to its ability to inhibit Bcl-2 and Bcl-XL and to suppress ERK activity in CDDP- or DXR-treated HER2-overexpressing cancer cells. T/t-common could enhance the antitumor activity of DXR on HER2-overexpressing ovarian tumor in NOD/SCID mice, suggesting that combination therapy using T/t-common and chemotherapeutic agents may provide a new approach for treating HER2-overexpressing cancers.

Targeted therapy in head/neck and gastric cancers

The epidermal growth factor receptor (EGFR/HER1) is a member of the erbB family of receptor tyrosine kinase proteins, which also includes HER2, HER3, and HER4. EGFR is almost universally expressed in squamous cell carcinoma of head and neck (SCCHN), and high levels of expression have been correlated with a poor clinical prognosis [1]. Cetuximab, an IgG1 monoclonal antibody against EGFR, has demonstrated improved survival and disease control when used in combination with radiation therapy for the treatment of locally advanced SCCHN, and in combination with platinum-based chemotherapy in recurrent or metastatic SCCHN [2-4]. Additionally, single-agent cetuximab is active and provides good disease control rate and duration in platinum-refractory SCCHN [5]. Comparison of cetuximab versus cisplatin concurrently with radiotherapy is under investigation in patients with human papillomavirus-associated oropharyngeal cancer, who have better prognosis and may benefit from less toxic treatment [6]. Overexpression of HER2 in gastric cancer results in aggressive clinical course and poor prognosis [7]. Trastuzumab, a monoclonal antibody against HER2, exhibits antitumor activity in HER2 overexpressed gastric cancer cells, and enhances effects of chemotherapy in gastric cancer xenograft overexpressing HER2 [8]. The ToGA study screened about 3,800 patients with advanced gastric cancer from 24 countries, and HER2 overexpression was detected in 22% [9]. Higher rates of HER2 overexpression occurred in intestinal and proximal or gastroesophageal junction cancers than in diffuse or distal gastric cancers. In TOGA study, 584 patients with HER2 overexpression were randomized to receive fluoropyrimidine and cisplatin treatment with or without trastuzumab. Patients who received trastuzumab plus chemotherapy achieved longer overall survival (13.8 months vs. 11.1 months, P= 0.0046), longer progression-free survival (6.7 months vs. 5.5 months, P=0.0002), and higher response rates (47% vs. 35%, P=0.0017) than those who received chemotherapy alone. Complete response was noted in 5.4% of patients receiving trastuzumab plus chemotherapy vs. 2.4% in chemotherapy alone. There were no significant differences in the toxicities between these two groups. This study has established a new paradigm using trastuzumab in combination with chemotherapy in patients with advanced gastric cancer overexpressing HER2. Neoadjuvant treatment is a standard of care for locally advanced esophageal and gastric cancer. We have previously reported a case of pathological complete response after neoadjuvant chemotherapy with trastuzumab-containing regimen in HER2-overexpressing gastric cancer [10]. Incorporating trastuzumab as a part of neoadjuvant therapy in esophageal and gastric adenocarcinoma overexpressing HER2 is currently under active investigation. Radiation Therapy Oncology Group is conducting a phase III neoadjuvant study in patients with HER2-overexpressing esophageal adenocarcinoma to determine if trastuzumab increases disease-free survival when added to chemoradiotherapy [11]. Other studies conducted in Europe are adding trastuzumab to oxaliplatin-based regimen as perioperative chemotherapy for HER2-overexpressing esophagogastric or gastric adenocarcinoma, and looking for improvement of pathological complete response and disease-free survival [12,13]. Pertuzumab is a monoclonal antibody interfering with HER2 dimerization with other HER receptors such as EGFR, HER3 and HER4. Pertuzumab and trastuzumab bind to HER2 at different sites, and combination of both antibodies leads to stronger inhibition of erbB signaling and greater therapeutic efficacy when combined with docetaxel in breast cancer [14]. Combination of pertuzumab and trastuzumab with platinum-based chemotherapy is currently studied in HER2-overexpressing gastric cancer [15].

Abstract 1684: The potential role of HER3 on HER2-driven biliary carcinogenesis and the possibility for targeted therapy with pertuzumab

Abstract HER2 has been implicated in biliary carcinogenesis, because gene amplification of HER2 has been observed in 20% of gallbladder carcinoma (GBC) (Kawamoto, et al. GCR 2007). The first-in-class HER2 dimerization inhibitor (HDI) pertuzumab is a humanized monoclonal antibody that binds HER2 at an epitope that prevents HER2 from dimerizing with ligand-activated HER family receptors, In this study, we determined the expression of HER family proteins in GBC, cholangiocellular carcinoma(CC), and examined whether treatment with pertuzumab would affect GBC and CC cell lines. Methods: HER family expression was examined in surgical or biopsied specimens from 47 GBCs and 57 CCs by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Using 6 GBC and 8 CC cell lines, heregulin-stimulated cell proliferation and its inhibition by pertuzumab, were tested in vitro by MTT assay. The phosphorylated HER family proteins and their respective downstream molecules were examined by Western blot analysis. Anti-tumor activity of pertuzumab in vivo was evaluated in the CC cell line KMCH-1 xenograft model with weekly intraperitoneal administration. Results: Over-expression of HER2 or HER3 was observed in 14-34% of all patients (Table 1). Six of 14 cell lines demonstrated over-expression of both HER2 and HER3, and heregulin stimulation increased the proliferation in 3 of them. In the HER2- and HER3- over-expressing KMCH-1, pertuzumab significantly inhibited cell proliferation in a dose-dependent manner. Pertuzumab completely blocked heregulin-induced phosphorylation of HER3. Furthermore, suppression of downstream pathway molecules including pAKT and pS6 were observed. The xenograft model also revealed tumor growth inhibition with pertuzumab. Conclusions: Pertuzumab demonstrated significant antitumor activities in HER2 and HER3 over-expressing cancer cells. Thus, pertuzumab may provide a new therapy option for both HER2- and HER3- positive GBC and CC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1684. doi:1538-7445.AM2012-1684

Abstract 5700: Preparation and evaluation of glycosylated paclitaxel loaded in tastuzumab-immunoliposome

Abstract Targeted drug delivery is considered to enhance the therapeutic effects of anti-cancer agents because high amount could be administered with least side effects. However, water-insoluble hydrophobic chemical compounds such as paclitaxel are not suitable for efficient drug delivery. We conferred water-solubility of paclitaxel by coupling glucose at 7-OH to produce 7-alpha-glucosyloxyacetylpaclitaxel (7-GLG-PT). Although the 7-GLG-PT showed lower cytotoxicity than paclitaxel, encapsulation of 7-GLG-PT into liposomes was more efficient than that of paclitaxel. In our experiment, paclitaxel encapsulated liposomes was not detectable. Trastuzumab was then conjugated on the surface of 7-GLG-PT liposomes to prepare immunoliposome (GLG-PT-IL). The GLG-PT-IL exhibited cytotoxicity on SK-BR-3 cells overexpressing HER2. The IC50 values of paclitaxel, 7-GLG-PT, 7-GLG-PT liposome and GLG-PT-IL were 6, 126, 130 and 94 nM, respectively. From this evaluation, the concentration of each formula to suppress the cell growth completely was estimated around 30 to 300 nM. At these concentrations, the time required for each formula to suppress the growth at the half maximal level was evaluated. GLG-PT-IL showed the shortest time of 5 hours, which was one third of the others when compared with paclitaxel, 7-GLG-PT and 7-GLG-PT liposome. The specific targeting potential of the immunoliposome on SK-BR-3 cells was assessed. The accumulation on the cell surface was competitively inhibited by the excess amount of free tastuzumab. The endocytosis of the immunoliposomes was simultaneously confirmed by time-lapse imaging. Thus GLG-PT-IL has been successfully demonstrated to target the HER2-overexpressing cancer cells specifically suppressing their growth. Trastumab itself is considered to induce the antibody dependent cellular cytotoxicity, so that the endocytosis of HER2 may not be always necessary. However, HER2-mediated endocytosis was induced by the immunoliposomes while single trastuzumab did not show the endocytosis in SK-BR-3 cells. In this context together with the results demonstrated in this study, the 7-GLG-PT immunoliposome should be a promising candidate of highly efficient drug delivery system of cancer treatment targeting HER2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5700. doi:1538-7445.AM2012-5700

Abstract C90: HER2-targeted liposomal doxorubicin MM-302 has a favorable cardiosafety profile in preclinical models.

Abstract Introduction: Anthracyclines have historically been the backbone of anticancer therapy for decades; however, the clinical observation of permanent cardiotoxicity has limited their broader use. In the HER2 positive breast cancer setting, the combination of trastuzumab with anthracyclines was associated with an unacceptable risk of clinical cardiotoxicity. This appears to be related to 1) doxorubicin-induced cardiomyocyte stress and 2) inability of the cardiomyocytes to respond to the stress due to the trastuzumab-related impairment of HER2 signaling. Encapsulation of doxorubicin into liposomes (Doxil®) has been associated with a reduced risk of doxorubicin-associated cardiotoxicity relative to free doxorubicin. MM-302 is a HER2-targeted liposomal doxorubicin-based agent designed to target doxorubicin to HER2-overexpressing cancer cells, while limiting uptake into non-target cells. Since HER2-mediated signaling plays an important role in cardiac repair, the purpose of this work was to support clinical development of MM-302 by investigating whether HER2-targeting of liposomal doxorubicin would adversely affect the cardiac safety profile relative to untargeted liposomal doxorubicin. Methods: MM-302, untargeted liposomal doxorubicin and free doxorubicin were compared for their uptake into HER2-overexpressing cancer cells and two models of human stem cell-derived cardiomyocytes. High-content microscopy was used to determine the effects of exposure to specific cellular targets in cardiomyocytes. Mouse biodistribution studies were used to assess the total and nuclear accumulation of doxorubicin in mouse cardiac tissue upon treatment with either MM-302, untargeted liposomes or free doxorubicin. Quantitative immunofluorescence was used to quantify the HER2 expression on human normal and diseased heart specimens. Kinetic computational modeling was applied to interpret study results and to make predictions on human heart exposure to doxorubicin based on the experimental mouse data. Results: Human stem cell-derived cardiomyocytes showed nuclear accumulation of doxorubicin followed by cell death upon free doxorubicin treatment but not upon MM-302 or untargeted liposomes treatment. Similarly, MM-302 resulted in a significantly lower nuclear accumulation of doxorubicin compared to free doxorubicin treatment in mouse cardiac tissue. HER2 expression levels on normal and diseased human heart tissue were shown to be ≤100,000 receptors/cell, in concordance with the HER2 levels on the stem cell-derived cardiomyocytes found in vitro. Computational modeling predicts liposomal encapsulation to significantly protect the human heart from exposure to doxorubicin, consistent with clinical findings. Conclusions: MM-302 is not taken up by human cardiomyocytes via HER2, but selectively increases doxorubicin delivery to human HER2-overexpressing tumor cells. MM-302 can potentially improve the clinical efficacy demonstrated by conventional anthracyclines and maintain the cardiac safety profile of untargeted liposomal doxorubicin in patients with HER2 overexpressing cancers. Clinical evaluation of this patient population is currently in Phase I. 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 C90.

Abstract A118: Development and evaluation of novel herceptin immunoliposome encapsulating cisplatin against HER2-overexpressing cancer cell.

Abstract Introduction: Liposomes, a number of designs have been applied to confer them specific affinity to tumor sites, are considered as appropriate carriers to delivery anti-cancer agents to tumor sites so as to reduce side effects. Breast cancer is one of disease with large numbers of patient all over the world. HER2/neu overexpressed breast cancer is malignant and poor prognostic. Targeting therapy of HER2 is required to overcome such a critical cancer. Herceptin (Trastuzumab) is antibody drug for HER2 overexpressed cancer. We exploited Herceptin to enhance targeting ability of liposome and anti-cancer activity by using cis-diamminedichloro-platinum (II) (CDDP) encapsulating liposome. We have previously developed efficient procedure of encapsulation of CDDP. Although it has long been a substantial problem to prepare liposomes loading high quantity of CDDP due to its insolubility in aqueous phase, our procedure enabled the loading efficiency hundreds more than those reported previously. In this study, these techniques were combined together to prepare liposomes conjugated with Herceptin loading CDDP to target breast cancer in vivo without unwanted side effects due to the toxicity of CDDP. Methods: Liposomes were prepared by improved cholate dialysis method. The lipids mixed in methanol/chloroform solvent were evaporated, followed by dissolved in solution of cis-diamminedinitratoplatinum (II) (CDDP3), one of the various CDDP derivatives with of high water solubility and is readily converted into CDDP in the presence of chloride ions, and then dialyzed against sodium chloride. Herceptin was conjugated with CDDP pre-loaded liposomes by cross-link agent DTSSP.CDDP encapsulated liposome and Herceptin conjugated immunoliposome were evaluated for anti-cancer activity in vitro by MTT assay. Targeting ability of immunoliposome was confirmed by confocal laser scanning and by in vivo imaging of BT474 xenografted model to confirm whether liposomes may accumulate at the tumor region. BT474 human ductal carcinoma was xenografted to estradiol capsule pre-implanted Balb/c (nu/nu) female mice. Drugs were administered to mice at dose of 20 mg/kg of mice body weight. The tumor sizes were monitored and survival rate of drug administered mice are calculated to evaluate whether drug administration contributed to prolong life of mice. Results: The peak sizes of liposomes were approximately 200 nm, whose sizes are suitable to accumulate in the tumor region via enhanced permeation and retention (EPR) effect. Though CDDP sensitivity of BT474 was less than other cell lines such as SKBr3, SKOv3 and MDA-MB-231, encapsulation of CDDP into liposome enhanced anti-cancer activity for BT474. Though fluorescence from liposomes encapsulated with Cy5.5 was detected from tumor of mice administered both normal liposome and Herceptin-liposome, mean tumor volume of the mice treated with Her-CDDP-Lip got smaller than that of the mice treated with CDDP-Lip. All mice treated with CDDP alone died within 4 days, while fifty percent of mice treated with Her-CDDP-Lip and CDDP-Lip survived up to the end of experiment. Conclusions: The Herceptin liposome was with strong anti-cancer activity both in vitro and in vivo when compared to usage of CDDP alone. We developed new combination therapy of Herceptin with CDDP, not common for treatment of breast cancer. 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 A118.