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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

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

Jang-Chang Leea,b, Li-Chen Choua, Jin-Chemg Liena, Jia-Chiun Wuc, Chi-Hung

Huangd, Chao-Ho Chunga, Fang-Yu Leee, Li-Jiau Huanga, Sheng-Chu Kuoa, f** and

Tzong-Der Wayc,g*

aGraduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China

Medical University, Taichung, Taiwan

bSchool of Pharmacy, College of Pharmacy, China Medical University, Taichung,

Taiwan

cDepartment of Biological Science and Technology, College of Life Sciences, China

Medical University, Taichung, Taiwan

dTaiwan Advance Biopharm, Inc., Xizhi City, Taipei, Taiwan

eYung-Shin Pharmaceutical Industry Co., Ltd. No. 1191, Sec. 1, Chung Shan Rd.,

Tachia, Taichung, Taiwan

fChinese Medicine Research and Development Center, China Medical University,

Taichung, Taiwan

gInstitute of Biochemistry, College of Life Science, National Chung Hsing University,

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Tzong-Der Way, Ph.D.

Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan

No.91 Hsueh-Shih Road, Taichung, Taiwan 40402 Tel: +886-4-2205-3366 ext: 5209

Fax: +886-4-2203-1075

E-mail: tdway@mail.cmu.edu.tw **Co-corresponding author:

Sheng-Chu Kuo, Ph.D.

Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, Taichung, Taiwan

No.91 Hsueh-Shih Road, Taichung, Taiwan 40402 Tel:(886)-4-2205-3366 ext: 5608

Fax: (886)-4-22030760

E-mail: sckuo@mail.cmu.edu.tw

Running title: CLC604 inhibits the HER2 expression

Abbreviations:

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CLC604, (1-benzyl-3-(p-hydroxymethylphenyl)-5-methylfuro[3,2-c]pyrazol); EGFR, epithelial growth factor receptor; HIF-1α, hypoxia-inducible factor-1α; Hsp90, 90-kDa heat shock protein; MG132, Z-Leu-Leu-Leu-al; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; YC-1, 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole

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Abstract

HER2 has become a solicitous therapeutic target in metastatic and clinical drug-resistance cancer. Here, we evaluated whether or not YC-1 and its furopyrazole and thienopyrazole analogues could repress the expression of HER2 protein. Among the test compounds, 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 suggest that CLC604 reduced HER2 expression through a posttranscriptional mechanism and involvement of proteasomal activity. CLC604 disrupted the association of Hsp90 with HER2 resulting from the inhibition of Hsp90 ATPase activity. Moreover, we found that CLC604 both significantly enhances the antitumor efficacy of clinical drugs on HER2-overexpressing tumors and efficiently reduces HER2-induced drug resistance in vitro and in vivo. These findings show that CLC604 should be developed further as a new antitumor drug candidate for treatment of drug-resistant cancer.

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Breast cancer and ovarian cancer remain major health problems, as well as the most common malignancies diagnosed in women (1). Approximately 30% of metastatic human breast cancer and ovarian cancer have been associated with the over-expression or amplification of HER2 receptor (2,3). HER2 is a 185 kDa transmembrane receptor with tyrosine kinase activity and may dimerize with the other epithelial growth factor receptor (EGFR) family members, including EGF receptors (HER1), HER3, and HER4. Overexpression of HER2 has been frequently found in various types of human cancers, such as breast, gastric, lung, ovarian, kidney, and bladder cancers. HER2 overexpression in cancer cells have been proved to enhance cell proliferation, increase tendency for metastasis, shorten disease-free survival, induce clinical drug-resistance, and lower overall survival rates (4,5).

HER2-mediated signaling has also been demonstrated to be involved in anti-apoptosis induced by certain proapoptotic stimuli (4). Moreover, previous studies have indicated that reducing the HER2 expression of cancer cells may attenuate its anti-apoptotic signaling and suppress HER2-mediated malignant phenotypes. Therefore, HER2 is not only a potent oncogene, but also an excellent therapeutic target in breast and ovarian cancers. Monoclonal antibodies were the first anti-HER2 strategy to be used for clinical therapy (6). Trastuzumab (Herceptin), a recombinant humanized monoclonal antibody, is used to treat metastatic breast cancer via directly

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counteracting the extracellular domain of HER2. Although Herceptin is known as a successful therapeutic antibody, only one-third of HER2-overexpressing metastatic breast cancers respond to Herceptin single agent, while almost two-thirds respond to combined Taxol-Herceptin regimens. However, these responses are short-lived, averaging less than one year (7), and the recurring resistance for Herceptin have been observed in the majority of patients within one year (8,9). Identification of the potential mechanisms of Herceptin-resistance can be extremely helpful for the development of new compounds that might overcome such resistance and demonstrate additive/synergistic antitumor effects when administered in association with Herceptin.

The 90-kDa heat shock protein (Hsp90) is a protein chaperone whose functions are to promote the maturation and conformational stabilization of a subset of cellular proteins, and it is crucial in signal transductions of cell proliferation and survival (10). During client processing, ATP binding to Hsp90 drives momentous conformational change in the chaperone and ultimately leads to ATP hydrolysis (11). HER2, a client protein of Hsp90, is known to interact with Hsp90 to acquire proper protein function; therefore, using inhibitors of Hsp90 to target HER2 through dissociation of HER2 from the chaperone leads to degradation of HER2 by a proteasome-dependent manner (11,12). The Hsp90 inhibitors such as geldanamycin and its less cytotoxic analogue,

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17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), through binding to an ATP pocket in the NH2-terminal domain of this protein, then inhibit the Hsp90 chaperone

function (11,13).

Numerous structure-activity relationships and biochemical assay data indicate that 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) have high potential as a new anticancer drug candidate (14,15). In vivo xenograft studies have also revealed that YC-1 exhibited exceptional anti-tumor activity against various cancer cell lines and prolonged the survival time of tumor-bearing mice, but without evident toxicities (16,17). The anti-cancer effect of YC-1 seemed to be the consequence of its multiple actions, including anti-inflammation activity, suppression of the hypoxia-inducible factor-1α (HIF-1α) expression, influence on the differentiation of stem cells, and promotion of the NK cell differentiation by activating the p38-MAPK pathway (18-20).

In our previous study, we described that YC-1 furopyrazoles and thienopyrazoles isosteric analogues exhibited greater cytotoxicity against HL-60 cells than YC-1, and their physiochemical properties and biological mechanisms appeared at variance to YC-1 (15,21). As part of our continuing search for potential anticancer drug candidates among YC-1 analogues, we investigated further the anticancer activity and biological mechanisms of various YC-1 isosteric analogues in vitro and in vivo. In this

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present study, eight furopyrazole and thienopyrazole compounds were chosen for evaluation of their effect on HER2 expression. Among them, CLC604 (1-benzyl-3-(p-hydroxymethylphenyl)-5-methylfuro[3,2-c]pyrazol) seemed more sensitive to HER2-overexpressing cancer cells. Thus, among the tested YC-1 analogues, CLC604 is considered the most promising compound for further study of physiochemical properties and biological mechanisms.

Combinative cancer therapy has become a common approach for increasing curative rates and decreasing side effects leading to improving the quality of life for patients. Consequently, we performed in vitro and in vivo synergistic treating for breast cancer cells with HER2 over-expression using the combination of CLC604 with low dose clinical drugs (Taxol, Doxorubicin, and Etoposide) in contrast with treating individually. We found that CLC604 might repress the growth of HER2-overexpressing breast tumors in MCF/Her18 tumor-bearing mice and increase the potency of Taxol.

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Materials and methods

Chemicals and reagents. YC-1, furopyrazole and thienopyrazole analogues of YC-1 were synthesized in our laboratory. Cell culture materials were obtained from Invitrogen (Burlington, Ontario, Canada). Antibodies and reagents were purchased from commercial sources: antibodies against Akt and c-Raf were purchased from Cell Signaling Technology (Beverly, MA); an antibody against Hsp90 was from BD Transduction Laboratory; antibodies against CDK4 and HER2 (9G6) were from Santa Cruz Biotechnology (Santa Cruz, CA); an antibody against HER2 (Ab3) was from Calbiochem company (San Diego, CA); anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase, a β-Actin antibody, an -tubulin antibody, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), actinomycin D, cycloheximide, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), Z-Leu-Leu-Leu-al (MG132), G418, Etoposide (E1383), Taxol (T7402), and Doxorubicin (D1515) were obtained from Sigma Chemical Co. (St. Louis, MO). Protein A/G-agarose was from an upstate company; Sea Plaque Agarose (low melting temperature agarose) was purchased from the Lonza Company.

Cell lines and cell cultures. The human breast and ovarian cancer cell lines used in this study were SKOV3, SKOV3.ip1, MDA-MB 453, and SKBr3, all of which overexpressing HER2, MCF-7, MDA-MB 231, and MDA-MB 435/neo all which

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express the basal level of HER2. The HBL-100 cell line, which is derived from a normal human breast tissue, was transformed by SV40 large T antigen and expresses a basal level of HER2. In addition, we used MCF-7/HER18 and MDA-MB 435/HER2; MCF-7 and MDA-MB 435 were stably transfected with pSV2/HER2 and HER2 over-expression. MDA-MB 453, MDA-MB 231, and MCF-7 were grown in DMEM supplemented with 10% fetal bovine serum, and SKBr3 was cultured in MyCoy’s 5A Medium (modified). Other cells were cultured in DMEM/F12 supplemented with 10% FBS. Cells were grown in a humidified incubator at 37°C under 5% CO2 in air.

Preparation of cell lysate, immunoblotting, and immunoprecipitation. Cells were treated with various agents, as indicated in figure legends. After treatment, cells were washed with cold PBS and lysed with a lysis buffer [1% Triton X-100, 10% glycerol, 10 µg/mL Leupeptin, 1 mmol/L sodium ortho-vanadate, 1 mmol/L EGTA, 10 mmol/L NaF, 1 mmol/L sodium pyrophosphate, 100 µmole/L β-Glycerophosphate, 20 mmol/L Tris-HCl (pH 7.9), 137 mmol/L NaCl, 5 mmol/L EDTA, and a protease inhibitor (1:10000)] in immune-blotting. For immune-precipitation, cells lysed with RIPA-B buffer [1% Triton X-100, 150 mmol/L NaCl, 20 µmol/L NaHPO4 (pH 7.4), 0.1

mmol/L sodium ortho-vanadate, 1 mol/L NaF, protease inhibitor (1:2500)]. 1 mg of each sample was mixed with 1 µg antibody and 50 µL Protein A-agarose at 4°C for 3

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h. The immune-precipitates were washed in RIPA-B buffer without a protease inhibitor, eluted with the SDS sample loading buffer, and processed for immune-blotting analyses, as described previously (22). For preparation of Triton X-100 soluble and insoluble fractions, cells were lysed with a lysis buffer containing 1% Triton X-100, as described above. After removal of Triton X-100 soluble cell lysate supernatants by centrifugation, the pellets were washed once with the lysis buffer and a 1x SDS loading buffer (50 µL) was then added to the pellets and heated at 95°C for 15 min to dissolve the Triton X-100-insoluble proteins.

Determination of cell viability by MTT assay. The effects of YC-1, furopyrazole and thienopyrazole analogues of YC-1, and three clinical drugs (Doxorubicin, Etoposide, and Taxol) on cell viability were examined by MTT assay. Cells were treated with various doses of the drugs for the indicated times, and the MTT dye was then added to each well. After 4 h incubation, the growth medium was removed and the formazan crystals, generated by oxidation of the MTT dye by cell mitochondria, were dissolved in 0.04 N HCl in isopropanol. The absorbance was measured at 570 nm, and the cell survival ratio was expressed as a percentage of the control viability (23).

Flow cytometry analysis. Cells were treated with various agents for the indicated times, harvested by trypsinization, fixed with 70% (v/v) ethanol at 4°C for 30 min, and washed twice with phosphate-buffered saline (PBS). After centrifugation, the

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cells were incubated with 0.1 mL of a phosphate-citric acid buffer [0.2 M NaHPO4

and 0.1 M citric acid (pH 7.8)] for 30 min at room temperature. The cells were then centrifuged and re-suspended in 0.5 mL PI solution comprising Triton X-100 (0.1%, v/v), RNase (100 mg/mL), and PI (80 mg/mL). The percentage was analyzed with FACScan and the Cell Quest software (Becton Dickinson; Mountain View, CA). Soft agar colony formation assay. The effects of CLC604 on the soft agar colony formation of various human breast cells were investigated. Briefly, cells (1 X 104)

were seeded in 6-cm culture dish containing 0.35% low-melting agarose over a 0.7% agarose layer in the presence of varying concentrations of CLC604 or a control vehicle and incubated for 3 weeks at 37°C. Colonies were stained with p-iodonitrotetrazolium violet (1 mg/mL), and those colonies larger than 100 μm were measured. The difference in effects of CLC604 between groups expressing an HER2 basal level and over-expression were evaluated using ANOVA.

Cell transfection. Plasmid pSV2-erbB2, a constitutive expression vector, carries the 4.4-kb full-length human HER2 cDNA under the control of the SV40 promoter/enhancer sequence. 6 x 105 cells were transfected with 5 μg of DNA

mediated by 21 μL of a Lipofectin reagent. Experiments were performed after transfection.

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six-well plates. Experiments were performed 24 h after cell attachment. Cells were fixed in PBS containing 4% Formaldehyde for 10–15 min at room temperature. Cells were rinsed with PBS 2–3 times followed by blocking with 5% BSA (Sigma; St. Louis, MO) for 30 min. Incubations were performed with primary antibodies diluted in a blocking buffer at 4°C overnight, after which cover slides were washed and incubated for 30 min with the fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (CHEMICON) diluted in a blocking buffer. Cover slides were washed and mounted. Fluorescence was visualized using a Nikon Optiphot-2 microscope.

In vitro Hsp90 assay. Hsp90 proteins expressed as His6-tagged fusions were purified as described previously using Talon metal-affinity chromatography, Q-sepharose ion-exchange, and Superdex 75, 200, or sephacryl 400HR gel-filtration chromatography. Proteins were concentrated in 20 mM Tris (pH 7.5) containing 0–25 mM NaCl, 1 mM EDTA, and 0.5 mM DTT. The Hsp90 ATPase assay was performed as previously described (24).

In vivo studies. The animals used in this study were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) following China Medical University Institutional Animals Ethics Committee clearance (99-151-N). Female BALB/c SCID mice (18 g - 20 g; 6~8 weeks of age) were purchased from the National Animal Center, Taipei, Taiwan, and maintained in pressurized ventilated

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cages according to institutional regulations. Each SCID mouse was subcutaneously inoculated in the right flank with 2 x 106 (A) MCF-7 cells, and (B) MCF-7/Her18

cells in 0.5 mL PBS via a 24-gauge needle. Growth of MCF-7 tumors was supplemented with 0.72 mg of 60-day release estrogen pellets (Innovative Research of America, Sarasota, FL) which were implanted subcutaneously on the back of the animals 24 h prior to cell inoculation. After the appearance of a 100-mm3 tumor

nodule, tumor-bearing mice of each (A) and (B) were randomly divided into five groups (n=6) for treatment with vehicle, administered by i.p. injection with Taxol (5 mg/kg), Taxol (5 mg/kg) combined with CLC604 (50 mg/kg) and CLC604 (50 mg/kg and 100 mg/kg, respectively) every 5 days each week for 4 consecutive weeks. The animals were weighed and the tumors were measured using calipers twice a week before, during, and after drug treatment. The tumor volume was calculated with the following formula: 1/2 (L × W2), where L is the length and W is the width of the

tumor. At the end of the experiments, the animals were euthanized with carbon dioxide followed by cervical dislocation.

Western blot analysis of expression of HER2 protein in vivo. Protein extracts were prepared by homogenizing tumor tissues obtained from vehicle-treated animals, CLC604 alone, Taxol alone, and combined CLC604 and Taxol treated mice with a lysis buffer [20 mM Na2PO4 (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% aprotinin,

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1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, 100 mM NaF, and 2 mM Na3VO4]. The protein content was determined against a standardized control using the

Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). A total of 50 μg of protein was processed for immune-blotting analyses as described previously (22). Statistical analysis. All values were expressed as mean ± SD. Each value is the mean of at least three separate experiments in each group. An ANOVA was used for statistical comparison. An asterisk (*) indicates that the values are significantly different from the control. (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

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Results

YC-1 and its furopyrazole and thienopyrazole isosteric analogues promote degradation of HER2. To investigate the effects of YC-1 and its furopyrazole and thienopyrazole isosteric analogues (Fig. 1A) on reducing the expression of HER2 protein, Western blotting was performed to establish the HER2 protein level in HER2-overexpressing human breast cancer MDA-MB 453 cells. These results showed that the expression of HER2 in MDA-MB 453 cancer cells was suppressed by YC-1 and its analogues in a dose-dependent manner (Fig. 1B). Moreover, we found that CLC609 and CLC604 exhibited more suppressing potency on the HER2 protein level than others did. Furthermore, we used the MTT growth assay to test the cytotoxicity of CLC609 and CLC604. Among them, CLC604 was found to be less cytotoxic to the immortalized noncancerous breast HBL-100 cell line (data not shown). Thus, CLC604 is considered the most promising compound for further studies. To assess the biological activity of CLC604 in terms of cell proliferation, cells were treated with CLC604 at different concentrations for 24 hours. The growth inhibition of the tested cell lines was in a dose-dependent manner, but to various extents (Fig. 1C). For example, CLC604 at 80 μM blocked >60% of growth in HER2-overexpressing cancer cells (SKOV3 and SKBr3). However, the inhibition was much less effective for those cells expressing a basal level of HER2 (MDA-MB 231,

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MCF-7, and HBL-100) under the same condition.

Effects of CLC604 on soft agar colony formation. Examining the effects of CLC604 on anchorage-independent growth is an important hallmark of the transformation phenotype. We seeded a variety of human cells into soft agar in the presence of a control vehicle or various concentrations of CLC604 and monitored them for colony formation. The colony-forming activity of HER2-overexpressing cancer cells (MDA-MB 453 and SKOV3.ip1) was more significantly suppressed than with HER2 basal level cells (MDA-MB 231 and HBL-100) at 80 μM CLC604 (Fig. 1D). The results suggest that CLC604 can reduce the HER2-mediated transformation phenotype of cancer cell lines. The above results indicated that CLC604 is safe and preferentially inhibits the growth of HER2-overexpressing cancer cell lines.

CLC604 preferentially inhibits the proliferation of HER2-overexpressing cancer cells. To evaluate the effects of CLC604 on cell proliferation, MCF-7 and MDA-MB 435 cells were stably transfected with pSV2-erbB2 and then treated with 80 μCLC604 for 24 and 48 h. The growth inhibition of these cell lines with CLC604 treatment were displayed in a time-dependent manner (Figs. 2A and 2B). Treatment with 80 μM CLC604 for 48 h inhibited over 40% of growth in HER2 overexpressing breast cancer cell lines which were stably expressing HER2 (MCF-7/HER18 and MDA-MB 435/HER2). However, the growth inhibition by CLC604 was much less in

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those parental cell lines expressing a basal level of HER2 (MCF-7 and MDA-MB 435). Conversely, CLC604 still significantly inhibited the HER2-overexpressing breast cancer cell line, which was stably expressing HER2 using cell anchorage-independent growth assay (Fig. 2C). Overall, these results suggest that CLC604 preferentially suppresses the growth of HER2-overexpressing cancer cells.

CLC604 changed the sub-cellular distribution of HER2. To confirm further the inhibitory effect on HER2 expression by CLC604, an immunofluorescence study with anti-HER2 antibody (Ab-3) showed that the control cells had strong immunofluorescence at the plasma membrane (Fig. 3A). However, after CLC604 treatment, the immunofluorescence at the plasma membrane disappeared and was replaced by diffused cytoplasmic punctate staining (Fig. 3B), which might be compatible with localization in the endoplasmic reticulum or the Golgi apparatus. Moreover, MDA-MB 453 was treated with 17AAG (inhibitor of Hsp90), the immunofluorescence also degraded (Fig. 3C), indicating that the stability of HER2 on plasma membranes may be associated with Hsp90.

HER2-mediated resistance to CLC604-induced apoptosis. Cells transiently transfected with a human cDNA encoding HER2 (pSV2-erbB2) recovered the immunofluorescence at the membrane (Fig. 3D). This phenomenon was not observed in cells transfected with a control vector (data not shown). In addition, as shown in

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Fig. 3G, pSV2-erbB2-transfected MDA-MB 453 cells demonstrated high resistance to CLC604-induced apoptosis, whereas the untransfected cells progressively underwent cell death. These results indicate that CLC604 reduced HER2 protein levels and induced apoptosis in the HER2-overexpressing breast cancer cells.

CLC604 inhibits HER2 expression by decreasing HER2 stability. To delineate more effectively the mechanism of CLC604-mediated HER2 down-regulation, we tested the effect of CLC604 on the HER2 protein level compared with the mRNA level. Combined with transcription inhibitor actinomycin D (AcD) or translation inhibitor cycloheximide (CHX), we examined the effect of CLC604 on the HER2 protein level in MDA-MB 453 breast cancer cells. HER2 protein level was detected by immunofluorescence assay and observed by confocal microscopy. An addition of CHX (Fig. 3E) or AcD (Fig. 3F) did not significantly alter the effect of CLC604 on the immunofluorescence pattern, indicating that CLC604 treatment did not alter HER2 mRNA levels or change the rate of de novo synthesis of HER2. To determine whether HER2 degradation is accelerated by CLC604, MDA-MB 453 cells were treated with CHX or with a combination of CHX and CLC604, and then the relative levels of HER2 protein in these cells were detected. As shown in Fig. 3H, the degradation rate of HER2 protein in MDA-MB 453 cells treated with CHX and CLC604 was faster than that of cells treated only with CHX. This indicates that a

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posttranslational mechanism contributed to CLC604-inducd HER2 depletion in HER2-overexpressing cancer cells. To investigate further the role of proteolysis in CLC604-mediated HER2 down-regulation, we conducted studies with the proteasome inhibitor MG132. In the absence of MG132, CLC604 reduced the protein levels of HER2 in both detergent (Triton soluble and detergent (Triton X-100)-insoluble cellular fractions. MG132 was found to inhibit CLC604-mediated reduction of HER2 levels in the Triton X-100-insoluble cellular fraction (Fig. 3I). These results suggest that proteasomal activity involves CLC604-induced HER2 degradation. Dissociation of HER2 from Hsp90 precedes the depletion of HER2. HER2 must be bound to the Hsp90 molecular chaperone complex, which is essential for HER2 stability and maturation (25). Hsp90 is an ATP-binding protein and has Mg2+

-dependent ATPase activity. To identify whether or not CLC604 disrupted the association of Hsp90 with HER2 resulting from competition with ATP, an in vitro Hsp90 ATPase activity assay was performed. Our results show that the efficacy of Hsp90 using ATP was inhibited by CLC604 treatment (IC50=14.32 μM  0.46). To

study further the mechanism of HER2 depletion by disassociating it with Hsp90, MDA-MB 453 cells were treated with either the control vehicle or 80 μM CLC604 at a variety of periods, and the binding of HER2 with Hsp90 were assessed. Equal amounts of fractionated proteins were immunoprecipitated with 1 μg of an anti-HER2

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monoclonal antibody, and the immunoprecipitates were blotted with HER2 and Hsp90 antibodies. The binding of HER2 with Hsp90 had already significantly decreased after CLC604 treatment (Fig. 3J). Moreover, Hsp90 inhibitor (17AAG), and CLC604 (40 μM and 80 μM) was also found to decrease the level of client protein of Hsp90 (HER2, Raf-1, AKT, CDK4) in MDA-MB 453 cells (Fig. 3K). CLC604 enhances the sensitivity of Doxorubicin, Etoposide, and Taxol on the growth of HER2-overexpressing cancer cells. To investigate whether or not CLC604 may sensitize HER2-overexpressing cancer cells to clinical drugs, we used MTT assay to investigate the effect of CLC604 treatment alone or in combination with Doxorubicin, Etoposide or Taxol on the growth of HER2-overexpressing cancer cells. To identify optimal conditions for combination treatment, we first examined the sensitivity of the HER2-overexpressing cancer cells to Doxorubicin, Etoposide or Taxol. As shown in Fig. 4, compared with cancer cells that expressed low levels of HER2, the HER2-overexpressing cancer cells demonstrated greater resistance to Doxorubicin (Fig. 4A), Etoposide (Fig. 4B) or Taxol (Fig. 4C). We then examined the combined effects of CLC604 and Doxorubicin, Etoposide or Taxol on the growth of MDA-MB 231 cells, which express low levels of HER2, and on the growth of SKBr3 cells, which overexpress HER2. The combination of CLC604 and Doxorubicin (Fig. 4A), Etoposide (Fig. 4B) or Taxol (Fig. 4C) synergistically inhibited HER2-overexpressing

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SKBr3 cells growth. However, no significant synergistic antiproliferative effect in the MDA-MB 231 cells exists.

To investigate specifically the effects of CLC604 on HER2-induced drug resistance, we required transformed cells whose drug resistance phenotypes are induced solely by an HER2 oncogene. To achieve this, we used MCF-7/HER18 breast cancer cell lines, which were stably expressing HER2. Next, we examined the effects of CLC604 on cell growth rate. As shown in Fig. 4D, the HER2-overexpressing cancer cells (MCF-7/HER18) were much more resistant to Taxol than parental breast cancer cells (MCF-7). In the efficacy of combinational conditions, the cytotoxicity of CLC604 combined with Taxol for HER2-overexpressing cancer cells were obviously more increased than when treated by CLC604 or Taxol along. These results indicate that CLC604 enhances the cytotoxicity effect of clinical drugs on HER2-overexpressing cancer cells and reduces the HER2-induced drug resistance of cancer cells.

CLC604 sensitizes HER2-overexpressing tumors in SCID mice to Taxol. As mentioned above, CLC604 acts synergistically with Taxol to inhibit the growth of HER2-overexpressing human breast cancer cells in vitro (Fig. 4D); we therefore examined whether CLC604 sensitizes HER2-overexpressing tumors in athymic SCID mice to Taxol. MCF-7 or MCF-7/HER18 cells were injected s.c. into athymic

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BALB/c SCID mice. When the solid tumors became palpable, mice were treated with either control, CLC604 alone (50 mg/kg and 100 mg/kg, respectively), Taxol alone (5 mg/kg), or a combination of CLC604 (50 mg/kg) and Taxol (5 mg/kg) given by i.p. injection every 5 days each week for 4 consecutive weeks. As shown in Fig. 5, treatment of the MCF7 tumor-bearing mice with Taxol alone significantly inhibited tumor growth and tumor weight (Figs. 5A and 5B). Moreover, the inhibitory effect on tumor growth was not enhanced by injecting CLC604 followed by Taxol (Figs. 5A and 5B). However, the MCF-7/HER18 tumor-bearing mice were much more resistant to Taxol than MCF-7 tumor-bearing mice (Figs. 6A and 6B). Interestingly, the combination treatment was significantly more effective than either of the treatments alone in MCF-7/HER18 tumor-bearing mice (Figs. 6A and 6B). During the antitumor activity evaluation, no apparent changes of mouse body weight were observed in either treatment or control groups (Figs. 5C and 6C). These in vivo experimental results unambiguously indicate that CLC604 significantly enhances the antitumor efficacy of Taxol on HER-overexpressing tumors and efficiently reduces the HER2-induced drug resistance.

CLC604 suppresses the expression of HER2 in vivo. To investigate whether or not repression of HER2 is connected with the therapeutic effects of CLC604 on tumors in vivo, MCF-7/HER18 tumors from the mice in each group (control, CLC604 alone (50

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mg/kg), Taxol alone, or CLC604 plus Taxol) was analyzed by Western blot analysis. HER2 levels in the CLC604-treated tumors were barely detectable, compared with levels in the control tumor; however, HER2 levels in the Taxol-treated tumor were not significantly changed (Fig. 6D). HER2 levels in the CLC604 combined with Taxol tumors were obviously more decreased than those treated with CLC604 or Taxol alone. These results are consistent with the concept that CLC604 suppresses the growth of HER2-overexpressing tumors in SCID mice by inhibiting the expression of HER2 and reduces the HER2-induced drug resistance of cancer cells.

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In the present study, we examined the relationship between the chemical structures and the inhibitory activity of YC-1 and its furopyrazole and thienopyrazole isosteric analogues on the expression of HER2 protein. We identified that one of the eight derivatives, CLC604, was more effective than other derivatives, even more than YC-1 in repressing the HER2 protein level. We have demonstrated that CLC604 preferentially inhibited the growth of original HER2-overexpressing cancer cells, but not the lines expressing basal levels of HER2, and suppressing transformation phenotype induced by the HER2 over-expression. Moreover, CLC604 was also found to be more effective in inhibiting the proliferation of HER2-overexpressing cancer cells, which were stably transfected by pSV2-erbB2, than expressing basal breast cancer cells.

Resent studies have demonstrated that constitutive phosphorylation of HER2 is associated with resistance to systemic therapies and local radiation therapies. Activation of HER2-containing heterodimers results in receptor autophosphorylation on COOH-terminal tyrosine residues, which become the docking sites for a number of signal transducers and adaptor molecules that initiate a plethora of signaling programs leading to cell proliferation, differentiation, migration, adhesion, protection from apoptosis, and transformation, among other effects. Our present study showed that HER2 is essential for cell survival. We treated original HER2-overexpressing breast

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cancer MDA-MB 453 cells with 80 μM CLC604 and the cell survival rate was detected by flow cytometry in comparison with MDA-MB 453 cells transiently transfected with pSV2-erbB2. These results demonstrated that pSV2-erbB2-transfected MDA-MB 453 cells exhibited high resistance to CLC604-induced apoptosis, whereas the untransfected cells progressively underwent cell death. This is consistent with a recent report that HER2-overexpressing cancer cells are dependent on HER2 levels for survival and, thus, are more sensitive to treatments that target HER2.

In previous studies, YC-1 has been identified as a novel class of NO-dependent stimulators of soluble gualylate cyclase (sGC) that exhibit therapeutic potential for the treatment of a range of vascular diseases, including hypertension, thrombosis, erectile dysfunction, and postangioplasty restenosis (14,26,27). Currently, YC-1 has been proved to suppress proliferation of HA22T via inhibiting CDK2 and CDK4 by upregulating p21CIP1/WAP1 and p27KIP1 in G

0-G1 arrest (28). Furthermore, YC-1 was

observed to suppress NF-κB activity via inhibiting the phosphorylation and degradation of I-κB and to induce apoptosis in prostate cancer cell line PC-3 cells (17). Moreover, YC-1 was recognized to inhibit angiogenesis via repressing VEGF by downregulating HIF-1 resulting in reducing cancer cell proliferation effectively (16,29-31). In the present study, our result showed that YC-1 could repress the protein

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level of HER2. Interestingly, we found that its isosteric analogue, CLC604, was more effective than YC-1 and its known derivatives in repressing the protein level of HER2. Moreover, CLC604 preferentially inhibited the growth of HER2-overexpressing cancer cells.

Hsp90 is required for refolding unfolded proteins and for cellular survival under environmental stress, and plays a key role in transducing proliferative and antiapoptotic signals in tumor cells especially; but in normal tissues, Hsp90 exists in a free, uncomplexed, or latent state (32). Consequently, inhibition of Hsp90 has emerged as a possible strategy for the treatment of advanced cancers (10). Recent studies have reported that the benzoquinone ansamycins such as geldanamycin and other Hsp90 inhibitors, such as 17-allyamino-17-demethoxygeldanamycin (17AAG), enhanced intracellular degradation of HER2 and involved targeting of the Hsp90 (13). Hsp90 forms complexes with HER2 and other client proteins. Recently, the mechanistic basis of Hsp90 client proteins sensitive to 17AAG has been demonstrated. The data of client protein half-life has showed that HER2 < mutant EGFR < Raf-1 < Akt < mutant BRAF< wild-type EGFR or HER2 is more sensitive to 17AAG than other client proteins (33). Once 17AAG blocks ATP binding to Hsp90, the chaperone complex associated with the client protein is biased toward a degradative fate, resulting in polyubiquitylation and subsequent destruction of the

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client. The mature HER2 requires Hsp90 association with its kinase domain to maintain the conformation necessary to heterodimerize with other ligand-activated HER proteins.

Our present study found that the HER2 protein level decreased faster in cells treated with CHX plus CLC604 than in cells treated with CHX alone. This result elucidated that a post-translational mechanism contributes to CLC604-induced HER2 instability and depletion in HER2-overexpressing cancer cells. We then attempted to identify whether or not CLC604 disrupted the association of Hsp90 with HER2 resulting from competition with ATP. Our in vitro Hsp90 ATPase activity assay showed that the efficacy of Hsp90 was inhibited by CLC604 treatment. CLC604 dissociated the complex between HER2 and Hsp90, and such dissociation precedes the depletion of mature HER2 at the plasma membrane. The depletion of mature membrane HER2 and the concomitant accumulation of HER2 in the cytoplasmic organelles are compatible with the notion that the complex of HER2 with Hsp90 is necessary for its maturation and subsequent transport to the plasma membrane. We thus hypothesized that CLC604 may also disrupt the association of HER2 and the chaperone complex through competition with ATP, and this may explain why CLC604 can deplete HER2 protein. Aside from HER2, other Hsp90 client proteins, such as Akt, c-Raf and cyclin-dependent kinase 4, were also reduced by CLC604.

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Recently, several studies have suggested that HER2-overexpressing cancer cells have developed drug resistant and relapsed capacity in treated with clinical drugs (34). All of these reports support the notion that HER2 overexpression is associated with chemoresistance. Moreover, data from clinical trials in breast cancer also suggest an association between HER2 overexpression and resistance to chemotherapy (35-38). Their results indicate that node-negative breast cancer patients whose tumor contains HER2 overexpression have a less favorable prognosis due to a lack of response to adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil-based chemotherapy. Later, a study of HER2 overexpression in epithelial ovarian cancer also demonstrated that patients whose tumors had the alteration were more likely to fail chemotherapy with cyclophosphamide and carboplatin (39). All of these reports support the notion that HER2 overexpression is associated with chemoresistance.

In the present study, we demonstrated that CLC604 is able to sensitize SKBr3 and MCF-7/HER18 breast cancer cells, which overexpress HER2 to in responding to the anticancer drugs Doxorubicin, Etoposide, and Taxol in vitro, but does not have the same effect in the MDA-MB 231 and MCF-7 cell lines, which express basal levels of HER2. These results suggest that HER2 is required for cell growth and Doxorubicin, Etoposide, and Taxol resistance; and that tumor repression by CLC604 alone and the synergistic effect of CLC604 plus Taxol on tumor growth in mice may be due to

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decreasing HER2. In the mice with HER2-overexpressing MCF-7/HER18 tumors, CLC604 significantly reduced tumor growth and weight. Western blot analysis indicated that the HER2 level was significantly reduced by CLC604 treatment compared with the control treatment. These results indicated that CLC604 indeed functions as an Hsp90 inhibitor and causes HER2-overexpressing cancer cells to be sensitive to Taxol in vivo. Our data corroborated recent reports that the more effective was obtained from combing 17AAG, an Hsp90 inhibitor, causing cancer cells to be sensitive to Taxol especially or other clinical drugs in treating HER2-overexpressing cancer (40).

Taken together, the central and novel findings in the present study are that: (a) CLC604 is more effective than YC-1 and its known derivatives; (b) CLC604 decreases the expression the level of HER2 in HER2-overexpressing cancer cells in vitro; (c) CLC604 significantly suppresses the growth of HER2-overexpressing cancer cells and transformed breast cancer cells in soft agarose; (d) CLC604 decreases the protein half-life of HER2 by proteasome activity; and (e) CLC604 may act as an Hsp90 inhibitor and cause HER2-overexpressing cancer cells to be sensitive to Taxol, Doxorubicin and Etoposide. The above findings may help improve the efficacy of preventive or therapeutic compounds against HER2-overexpressing cancer cells.

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Acknowledgment:

The investigation was supported by a research grants from the National Science Council (NSC-97-2320-B-039-008-MY3 and NSC-97-2320-B-039-003-MY3) to S.C. Kuo. Thanks are also due to support (in part) by the grant from the Department of Health (Taiwan), China Medical University Hospital Cancer Research Center of Excellence (DOH100-TD-C-111-005) and grant from China Medical University (CMU99-S-34 and CMU99-TC-02). We thank Prof. Mien-Chie Hung (The University of Texas M. D. Anderson Cancer Center, Houston, Texas) for generously providing cancer cell lines MDA-MB 453/neo, MDA-MB 435/HER2 and MCF-7/HER18.

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Figure legends

Figure 1. Effects of YC-1 and its analogues on the expression of HER2 protein. (A) Chemical structures of YC-1 and its furopyrazole and thienopyrazole isosteric analogues. (B) HER2-overexpressing breast cancer cells MDA-MB 453 were treated with YC-1 and its analogues (40 and 80 μM) at 37°C for 24 h. Immunoblotting was used to measure HER2 and β-actin. Change in the protein expression of the bands normalized to β-actin. Columns, mean of three independent experiments; bars, SD. (C) Cell viability was determined by MTT assays after continuous exposure to different concentrations of CLC604 at 37°C for 24 h. The number of viable cells after

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three independent experiments; bars, SD. (D) Effect of CLC604 on anchorage-independent growth of cancer cells. Cells (1x104 cells/well) were seeded in 6 cm dish

in culture medium containing 0.35% low-melting agarose over a 0.7% agarose layer in the presence of 80 μM of CLC604 or control vehicle and were incubated for 3 weeks at 37°C. Colonies larger than 100 μm were counted. The percentage of colony formation was calculated by defining the number of colonies in the absence of CLC604 as 100%. Columns, mean of three independent experiments; bars, SD. Figure 2. Effects of CLC604 inhibit the proliferation of HER2-overexpressing cancer cells. (A) MCF-7 and MCF-7/HER18 (B) MDA-MB 435/neo and MDA-MB 435/HER2 treatment with 80 μM CLC604 at 37°C for 24 h and 48 h, the effect on cell growth was examined by MTT assay. The number of viable cells after treatment was expressed as a percentage of the vehicle-only control. (C) Effect of CLC604 on anchorage-independent growth of breast cancer cells. Column, mean of three independent experiments. Bars represent the SD. Asterisk, values significantly different from the control. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3. HER2-mediated resistance to CLC604-induced apoptosis. MDA-MB 453 cells grown on coverslips were treated with (A) control vehicle, (B) 80 μM CLC604, (C) 17AAG (10 μM) and for 24 h. (D) Cells transfected with pSV2-erbB2 and incubated with 80 μM CLC604 for 24 h. (E) Prior to adding 80 μM CLC604, cells

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were pretreated with cycloheximide 20 g/mL or (F) actinomycin D 5 μM. Cells were fixed with 4% paraformaldehyde and stained with an HER2 antibody followed by a fluorescein isothiocyanate-conjugated secondary antibody (green), DPAI (blue). Analysis of subcellular distribution was performed by confocal microscopy. (G)Cells transfected with an empty vector or pSV2-erbB2 and treated with DMSO (Con) or CLC604 (80 μM) for 24 h and analyzed by flow cytometry as described under “Experimental Procedures.” (H)MDA-MB 453 cells were cultured with 20 μg/mL cycloheximide (CHX) in the presence or absence of 80 μM CLC604 for the indicated times. Top, representative experiment in which β-actin and  HER2 protein levels were assessed by Western blot analysis; bottom, quantification of HER2 expression normalized to the level of β-actin control. HER2 expression at the 0-h time point was set as 100%. (I) MDA-MB 453 cells were pretreated with MG132 (20 μM) for 30 min followed by 80 μM CLC604 for 9 h, and Triton X-100–soluble and Triton X-100– insoluble cell lysates were prepared and assessed by Western blotting with antibodies to HER2 and β-actin. (J) Dissociation of Hsp90-HER2 complex by CLC604. SKOV3.ip1 cells were treated with 80 μM CLC604 for the duration indicated. Cell lysates were immunoprecipitated with a mouse monoclonal anti-HER2 antibody and immunoblotted for HER2 and Hsp90. (K) CLC604 induced the degradation of the client protein of Hsp90 in MDA-MB 453 breast cancer cells. MDA-MB 453 cells

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were treated with the Hsp90 inhibitor 17AAG (10 μM) or CLC604 (40 and 80 μM) at 37℃ for 48h. Levels of HER2, Raf-1, AKT, CDK4, and β-actin were analyzed by Western blotting. Western blot data presented are representative of those obtained in at least three separate experiments.

Figure 4. Effect of clinical drugs (Doxorubicin, Etoposide and Taxol) alone or in combination with CLC604 on the proliferation of human breast cancer cells. MDA-MB 231 and SKBr3 cells were treated with 40 μM CLC604 alone or in combination with (A) 2.5 μM Doxorubicin, (B) 20 μM Etoposide, or (C) 400 nM Taxol at 37°C for 24h. MCF-7 and MCF-7/HER18 cells were treated with 40 μM CLC604 alone or in combination with (D) 400 nM Taxol at 37°C for 24 h. The effects on cell growth were examined by the MTT assay, and the percentage of cell proliferation was calculated by defining the absorption of cells not treated with drugs as 100%. The inhibitory effect was calculated as 100% minus the percentage of cell proliferation. Results are given as means; bars, SD.

Figure 5. Effect of CLC604 alone or in combination with Taxol in BALB/c SCID mice subcutaneous xenograft model. Female BALB/c SCID mice (n = 6) were subcutaneously inoculated with 2 x 106 MCF-7 cells. When the solid tumors were

palpable and tumor size was measured as shown at day 0, the mice were administered either a placebo, CLC604 (50 mg/kg and 100 mg/kg, respectively), Taxol (5 mg/kg),

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or CLC604 (50 mg/kg) plus Taxol (5 mg/kg) by i.p. injection every 5 days each week for 4 consecutive weeks. (A) Tumor volume (mm3), (B) tumor weight (g), (C) body

weight (g). Results are given as means; bars, SD.

Figure 6. Effect of CLC604 alone or in combination with Taxol in HER2-overexpressing BALB/c SCID mice subcutaneous xenograft model. Female BALB/c SCID mice (n=6) were subcutaneously inoculated with 2 x 106 MCF-7/HER18 cells.

When the solid tumors were palpable and tumor size was measured as shown at day 0, the mice were given either a placebo, CLC604 (50 mg/kg and 100 mg/kg, respectively), Taxol (5 mg/kg), or CLC604 (50 mg/kg) plus Taxol (5 mg/kg) by i.p. injection every 5 days each week for 4 consecutive weeks. (A) Tumor volume (mm3),

(B) tumor weight (g), (C) body weight (g). Results are given as means; bars, SD. (D) Western blot analysis of levels of HER2 protein in vivo. Protein extracts were prepared by homogenizing tumor tissues obtained from the control, CLC604-treated, Taxol-treated, and combined CLC604 and Taxol-treated mice with lysis buffer. Western blotting was conducted using an anti-HER2 antibody or an anti-β-actin antibody, as described in “Materials and Methods.”

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