HDAC inhibitor SNDX-275 enhances efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to overcome trastuzumab resistance

Cancer Letters 307 (2011) 72–79

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

HDAC inhibitor SNDX-275 enhances efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to overcome trastuzumab resistance Xiaoping Huang a,1, Shuiliang Wang a, Choon-Kee Lee b, XiaoHe Yang c, Bolin Liu a,⇑ a

Department of Pathology, University of Colorado Denver School of Medicine, Aurora, CO, United States The Myeloma and Amyloidosis Program, Department of Medicine, University of Colorado Denver School of Medicine, Aurora, CO, United States c Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States b

a r t i c l e

i n f o

Article history: Received 9 February 2011 Received in revised form 17 March 2011 Accepted 20 March 2011

Keywords: HDAC SNDX-275 erbB receptor Trastuzumab resistance Breast cancer

a b s t r a c t Trastuzumab (or Herceptin), as the first erbB2-targeted therapy, has been successfully used to treat breast cancer patients with erbB2-overexpressing tumors. However, resistances to trastuzumab frequently occur, and novel strategies/agents are urgently needed to abrogate the resistant phenotype. Our current study explores the potential of SNDX-275, a class I HDAC inhibitor, to overcome trastuzumab resistance and investigates the combinational effects of SNDX-275 and trastuzumab on both sensitive and resistant breast cancer cells. Cell proliferation assays showed that SNDX-275 significantly enhanced trastuzumabinduced growth inhibition in trastuzumab-sensitive, erbB2-overexpressing breast cancer cells. Importantly, SNDX-275 at its therapeutic range re-sensitized trastuzumab-resistant cells to trastuzumab-mediated growth inhibition. SNDX-275 in combination with trastuzumab resulted in a dramatic reduction of erbB3 and its phosphorylation (P-erbB3), and inhibition of Akt signaling. Apoptotic-ELISA and western blot analyses confirmed that the combinations of SNDX-275 and trastuzumab as compared to SNDX-275 alone significantly enhanced DNA fragmentation and induced more PARP cleavage and caspase-3 activation in both trastuzumab-sensitive and -resistant breast cancer cells. Furthermore, co-immunoprecipitation assays revealed that SNDX-275 mainly attenuated the interactions of erbB2 and erbB3 receptors, but had no significant effect on erbB2/IGF-1R or erbB3/IGF-1R associations in the trastuzumab-resistant breast cancer cells. These data indicated that SNDX-275 enhanced trastuzumab efficacy against erbB2-overexpressing breast cancer cells, and exhibited potential to overcome trastuzumab resistance via disrupting erbB2/erbB3 interactions and inactivating PI-3K/Akt signaling. SNDX-275 may be included in erbB2-targeted regimen as a novel strategy to treat breast cancer patients whose tumors overexpress erbB2. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: RTK, receptor tyrosine kinase; FDA, Food and Drug Administration; HDAC, histone deacetylase; HDACi, inhibitor of HDAC; EGFR, epidermal growth factor receptor; IGF-1R, insulin-like growth factor-1 receptor; MAPK, mitogen-activated protein kinase; PI-3K, phosphoinositide 3kinase; MTD, maximum-tolerated dose; ELISA, enzyme-linked immunosorbent assay; PTEN, phosphatase and tensin homolog; SNDX-275, N-(2aminophenyl)-4-[N-(pyridine-3-ylmethoxycarbonyl)aminomethyl]benzamide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt. ⇑ Corresponding author. Address: Department of Pathology, University of Colorado Denver School of Medicine, MS-8104, P.O. Box 6511, 12801 E. 17[th] Ave., Aurora, CO 80045, United States. Tel.: +1 303 724 3749; fax: +1 303 724 3712. E-mail address: [email protected] (B. Liu). 1 Present address: Department of Microbiology, University of Virginia, Old Medical School Room 4813, P.O. Box 800394, 21 Hospital Drive, Charlottesville, VA 22908, United States. 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.03.019

X. Huang et al. / Cancer Letters 307 (2011) 72–79

1. Introduction Gene amplification and/or overexpression of erbB2 occur in 25–30% of invasive breast carcinomas, and are significantly associated with a worse prognosis in breast cancer patients [1,2]. Recent advances in our understanding of the biology of breast cancer have led to improved patient survival with target therapies. Trastuzumab (or Herceptin), a humanized monoclonal antibody against erbB2 receptor, was the first erbB2-targeted therapy approved by FDA [3]. Although trastuzumab has been successfully used in early-stage and metastatic breast cancer therapy of patients with erbB2-overexpressing tumors as monotherapy [4] and in combination with other agents [5,6], not all breast cancer patients whose tumors overexpress erbB2 respond to trastuzumab and majority of patients who achieve an initial response to trastuzumabbased regimens develop resistance within one year [7,8]. These data indicate that both primary and acquired resistances to trastuzumab are common, and currently represent a significant clinical problem. It is critical to explore novel agents and/or strategies with therapeutic potential to overcome trastuzumab resistance against the aggressive erbB2-overexpressing breast cancers. Numerous studies indicate that deregulation of acetylation and deacetylation plays an important role in aberrant gene expression in human cancers [9,10]. Histone deacetylases (HDAC) have thus become attractive therapeutic targets, and many inhibitors of HDACs are actively under investigation as potential anti-cancer agents [11,12]. Indeed, HDAC inhibitors (HDACi) have been found to possess anti-cancer activity in a variety of tumor cell models via influencing cell cycle progression, apoptosis, differentiation, and tumor angiogenesis [13,14]. SNDX-275 (formerly known as MS-275, EntinostatÒ, Syndax Pharmaceuticals, Inc., Waltham, MA) is a synthetic benzamide derivative class I HDACi. It inhibits cancer cell growth with an IC50 in the submicromolar range, and exhibits both in vitro and in vivo activities against various human cancers [15– 17]. In the model of breast cancers, SNDX-275 has been shown to inhibit cell proliferation and/or promote apoptosis via different molecular mechanisms [18–22]. Currently, there are two clinical trials involving SNDX-275 in estrogen receptor (ER) positive breast cancer patients.2 We have recently studied whether SNDX-275 holds specific activities against certain subtypes of human breast cancers, with a focus to explore SNDX-275’s potential targeting erbB receptors, such as EGFR, erbB2, and erbB3, in breast cancer cells. Our data indicate that SNDX-275 is more effective at inhibiting cell proliferation and inducing apoptosis in erbB2-overexpressing than in basal-type breast cancer cells. Particularly, SNDX-275 downregulates the expression levels of erbB2 and erbB3, but not EGFR, and mainly inactivates the phosphoinositide 3-kinase (PI3K)/Akt signaling [23]. Coexpression of erbB3 and erbB2 is frequently observed in breast cancers [24] and cell lines [25], and erbB3 plays a critical role in breast cancer development driven by erbB2 amplification/overexpression [26]. Furthermore, activation

2

http://www.clinicaltrials.gov/ct2/results?term = SNDX-275.

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of the PI-3K/Akt signaling has been identified as the major determinant of trastuzumab resistance [27]. Indeed, our recent studies with a unique trastuzumab-resistant breast cancer model demonstrate that the erbB3 receptor interacts with both erbB2 and the insulin-like growth factor-1 receptor (IGF-1R) to form a heterotrimeric complex, which induces PI-3K/Akt activation and subsequently leads to trastuzumab resistance [28]. We hypothesized that SNDX-275 may be able to overcome trastuzumab resistance and enhance the efficacy of trastuzumab against erbB2-overexpressing breast cancer cells via downregulation of erbB3 expression and inactivation of the PI-3K/Akt signaling. In the current studies, we have investigated the combinational effects of SNDX-275 and trastuzumab on inducing growth inhibition and/or apoptosis in both trastuzumab-sensitive and -resistant breast cancer cells. 2. Materials and methods 2.1. Reagents and antibodies SNDX-275 (N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxycarbonyl) aminomethyl] benzamide) was provided by Syndax Pharmaceuticals, Inc., dissolved in DMSO to make a 20 mmol/L stock solution, and stored at 20 °C. Trastuzumab (HerceptinÒ, Genentech, Inc.) was obtained from University of Colorado Hospital pharmacy. Antibodies were obtained as follows: erbB2 (EMD Chemicals, Inc.); erbB3 and P-erbB2 (LabVision Corp.); P-erbB3, caspase-3, P-MAPK, MAPK, P-Akt (Ser473), Akt, Acetyl-Histone H3, Histone H3 (Cell Signaling Technology, Inc.); p27kip1 (Santa Cruz Biotechnology, Inc.); poly ADP ribose polymerase (PARP) monoclonal antibody (BIOMOL Research Laboratories, Inc.); and b-actin (Sigma Co.). All other reagents were purchased from Sigma unless otherwise specified. 2.2. Cells and cell culture Human breast cancer cell lines SKBR3, BT474, and MDAMB-453 were obtained from the American Type Culture Collection and maintained in DMEM/F-12 (1:1) medium (Sigma) containing 10% fetal bovine serum (Sigma). All three cell lines and the trastuzumab-resistant sublines, SKBR3-pool2 and BT474-HR20 [28], were cultured in a 37 °C humidified atmosphere containing 95% air and 5% CO2 and split twice a week. The identity of all the cell lines was recently confirmed with DNA profiling by University of Colorado Cancer Center’s DNA Sequencing & Analysis Core facility. 2.3. Cell proliferation assay The CellTiter96 AQ nonradioactive cell proliferation kit (Promega Corp.) was used to determine cell viability as previously described [23,28,29]. Briefly, cells were plated onto 96-well plates for 24 h, and then grown in either DMEM/F12 medium with 0.5% FBS as control, or the same medium containing different concentrations of SNDX-275 and/or trastuzumab incubated for another 72 h. After

X. Huang et al. / Cancer Letters 307 (2011) 72–79

2.5. Quantification of apoptosis An apoptosis ELISA kit (Roche Diagnostics) was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) as previously reported [23,28,29]. 2.6. Statistical analysis Statistical analyses of the experimental data were performed using a two-sided Student’s t test. Significance was set at a P value of <0.05. 3. Results

MDA MB 453 MDA-MB-453

P=0.003 P<0.0001 P=0.0002 P<0.0001 P=0.0005

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Immunoprecipitation and Western blot assays were performed as described [23,28,29]. Briefly, equal amounts of cell lysates were incubated with primary antibody for 2 h at 4 °C followed by incubation with protein A or G-agarose (Roche Diagnostics) at 4 °C overnight. The immunoprecipitates or equal amounts of cell lysates were boiled in SDS sample buffer, resolved by SDS–PAGE, transferred to nitrocellulose (Bio-Rad), and probed with primary antibody. After the blots were incubated with horseradish peroxidase-labeled secondary antibody, the signals were detected using the enhanced chemiluminescence reagents (Amersham Life Science).

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reading all wells at 490 nm with a microplate reader, the percentages of surviving cells from each group relative to controls, defined as 100% survival, were determined by reduction of MTS.

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Fig. 1. Trastuzumab and SNDX-275 inhibited cell proliferation of erbB2overexpressing breast cancer cells. BT474 (A) and MDA-MB-453 (B) human breast cancer cells were plated onto 96-well plates and incubated at 37 °C with 5% CO2. After 24 h, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing either trastuzumab (137 nmol/L) or the indicated concentrations of SNDX-275 alone or combinations of trastuzumab (137 nmol/L) and SNDX-275 for another 72 h incubation. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars, SD. Data shows a representative of at least three independent experiments.

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3.1. SNDX-275 enhances trastuzumab-induced growth inhibition of erbB2overexpressing breast cancer cells correlated with reduction of P-erbB3 and inactivation of Akt signaling

P-erbB3 erbB3 P-Akt

Our previous studies indicated that SNDX-275 preferentially inhibited cell proliferation/survival of erbB2-overexpressing breast cancer cells [23]. To explore whether SNDX-275 may enhance the efficacy of trastuzumab against erbB2-overexpressing breast cancers, we have investigated the anti-proliferation/anti-survival activities of combined use of SNDX-275 and trastuzumab in erbB2-overexpressing breast cancer cells (SKBR3, BT474, and MDA-MB-453). At its clinically achievable range (61 lmol/L), SNDX-275 significantly increased trastuzumab-mediated growth inhibition in BT474, MDA-MB-453, and SKBR3 cells (Fig. 1A and Fig. S1A). To closely imitate the clinical conditions and reduce the side effects of the drugs, we used two treatment regimens with SNDX-275 at the lower doses (0.2 lmol/L or 0.5 lmol/L) to study the combinational effects of SNDX-275 and trastuzumab on erbB2/erbB3 tyrosine kinase activities and their downstream signaling kinases Akt and MAPK. When the erbB2-overexpressing breast cancer cells were treated with trastuzumab (137 nmol/L) and/or SNDX-275 (0.5 lmol/L) for two days, trastuzumab alone mainly reduced the phosphorylation levels of erbB3 (P-erbB3) and Akt (P-Akt), whereas SNDX-275 alone had little inhibition on P-Akt in both BT474 and MDA-MB-453 cells (Fig. 2A). However, the combinations of trastuzumab and SNDX-275 significantly decreased the levels of P-erbB2 (BT474), P-erbB3 and P-Akt in both BT474 and MDA-MB-453 cells. Either trastuzumab or SNDX-275 alone or their combinations had little or no effect on P-erbB2 and P-MAPK (Fig. 2A). Similar effects of trastuzumab and/or SNDX-275 on P-erbB2, P-erbB3, P-Akt, and P-MAPK were also observed in SKBR3 cells (Fig. S1B). In the second treatment regimen, we further reduced the concentrations of trastuzumab (68.5 nmol/L) and SNDX-275 (0.2 lmol/L) to test their inhibitory effects on BT474 and MDA-

Akt P MAPK P-MAPK MAPK Ac-H H3 Histone H3 β-actin

Fig. 2. Combinations of trastuzumab and SNDX-275 significantly inactivated the erbB2/erbB3 receptors and their downstream signaling pathways in erbB2-overexpressing breast cancer cells. A and B, BT474 or MDA-MB-453 cells were treated with either trastuzumab, or SNDX-275 alone, or combinations of trastuzumab and SNDX-275 for 2 (A) or 4 (B) days. Cells were collected and subjected to western blot analyses with specific antibodies directed against erbB2, P-erbB2, erbB3, P-erbB3, P-Akt, Akt, P-MAPK, MAPK, histone H3, acetylated histone H3 (Ac-H H3), or bactin.

MB-453 cells in a 4-day interval (Fig. 2B). Similarly, the combinations of trastuzumab and SNDX-275 showed greater activities than either agent alone to reduce the levels of P-erbB3 and P-Akt. This longer time treatment with two agents together also significantly decreased the protein levels of both erbB2 and erbB3. While P-MAPK levels in BT474 cells were dramatically reduced upon trastuzumab and SNDX-275 treatment for

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B SNDX-275 PARP Pro-Casp-3 Cleaved Casp-3

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Fig. 3. Trastuzumab accelerates SNDX-275-induced apoptosis in erbB2-overexpressing breast cancer cells. A and B, BT474 or MDA-MB-453 cells were treated with either trastuzumab (137 nmol/L), or SNDX-275 (0.5 lmol/L) alone, or combinations of trastuzumab and SNDX-275 for 2 days. Cells were collected and subjected to western blot analyses with specific antibodies directed against PARP, Caspase 3 or b-actin (A), or apoptotic-ELISA (B). C and D, BT474 or MDA-MB-453 cells were treated with either trastuzumab (68.5 nmol/L), or SNDX-275 (0.2 lmol/L) alone, or combinations of trastuzumab and SNDX-275 for 4 days. Cells were collected and subjected to western blot analyses with specific antibodies directed against PARP, Caspase-3 or b-actin (C), or apoptotic-ELISA (D). Bars, SD. 4 days, the levels of P-MAPK in MDA-MB-453 cells still remained unchanged (Fig. 2B). Collectively, these data suggest that SNDX-275 enhances trastuzumab-induced growth inhibition in erbB2-overexpressing breast cancer cells mainly through reduction of P-erbB3 and inactivation of Akt. 3.2. SNDX-275 in combination with trastuzumab promotes apoptosis in erbB2-overexpressing breast cancer cells To study the molecular mechanism by which SNDX-275 enhances trastuzumab efficacy on inhibition of cell proliferation and/or survival, we investigated the effects of SNDX-275 and/or trastuzumab on induction of apoptotic cell death in erbB2-overexpressing breast cancer cells. Treatment with trastuzumab (137 nmol/L) or SNDX-275 (0.5 lmol/L) alone for 2 days had little effect on PARP cleavage and activation of caspase-3. In contrast, the combinations of trastuzumab (137 nmol/L) and SNDX-275 (0.5 lmol/L) clearly induced cleavages of both PARP and caspase-3, the hallmarks of caspase-dependent apoptosis, in BT474 and MDA-MB-453 cells (Fig. 3A). A specific ELISA assay measuring DNA fragmentation further verified that BT474 and MDA-MB-453 cells indeed underwent apoptosis upon combinational treatment with SNDX-275 and trastuzumab for 2 days (Fig. 3B). We next treated the same cells for 4 days with lower concentrations of trastuzumab (68.5 nmol/L) and/or SNDX-275 (0.2 lmol/L). Although treatment with either trastuzumab or SNDX-275 alone resulted in a clear PARP cleavage, their combinations significantly increased PARP cleavage and activation of caspase-3 in both BT474 and MDA-MB-453 cells (Fig. 3C). These results were also confirmed with the apoptosis specific ELISA (Fig. 3D). Taken together, our studies indicate that the combinations of SNDX-275 and trastuzumab induced caspase-dependent apoptosis in erbB2-overexpressing breast cancer cells.

3.3. SNDX-275 re-sensitizes trastuzumab-resistant breast cancer cells to trastuzumab-induced growth inhibition and apoptosis In order to explore whether SNDX-275 holds potential to overcome trastuzumab resistance in breast cancer therapy, we took advantage of two trastuzumab-resistant breast cancer cell lines BT474-HR20 and SKBR3-pool2 derived from BT474 and SKBR3 cell lines, respectively [28]. In contrast to the parental BT474 cells, BT474-HR20 cells were resistant to the treatment of either trastuzumab or SNDX-275 alone. However, the combinations of these two agents significantly inhibited proliferation/ survival of BT474-HR20 cells (Fig. 4A). Interestingly, although SKBR3pool2 cells showed no response to trastuzumab, it appeared these cells were still sensitive to SNDX-275 treatment. Nonetheless, SNDX-275 in combination with trastuzumab displayed a much greater inhibitory effect on SKBR3-pool2 cells (Fig. S2). These data suggest that SNDX-275 might reverse trastuzumab-resistant phenotype in vitro. We then investigated the combinational effects of SNDX-275 and trastuzumab on erbB2/erbB3 kinase activity and their downstream signaling in trastuzumab-resistant breast cancer cells. Similar results were obtained as we observed with the parental BT474 cells (Fig. 2), the combinations of SNDX-275 and trastuzumab mainly exhibited a profound activity on reduction of P-erbB3 and P-Akt levels in BT474-HR20 cells, although P-MAPK levels were also significantly decreased upon 4-day treatment with lower concentrations of trastuzumab and SNDX-275 (Fig. 4B). Moreover, western blot analyses and specific apoptotic-ELISA demonstrated that the combinations of trastuzumab and SNDX-275 induced PARP cleavage and caspase-3 activation (Fig. 5A), and promoted more DNA fragmentation (Fig. 5B) in BT474HR20 cells, regardless of 2-day or 4-day treatment regimen. These data indicate that SNDX-275 is able to enhance trastuzumab-mediated growth inhibition and apoptosis in trastuzumab-resistant breast cancer cells.

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A

A

* P< 0.001 vs single agent

120

80

*

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(Trastuzumab 68.5nmol/L SNDX-275 0.2μmol/L)

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2 days treatment

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(Trastuzumab 68.5nmol/L SNDX 275 0 0.2μmol/L) SNDX-275 2μmol/L)

+ -

+

+ +

Trastuzumab SNDX-275

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P-erbB2 erbB2 P-erbB3 erbB3

Apopto A osis (O OD@40 05 nm))

B

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2d days ttreatment t t

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(Trastuzumab 68.5nmol/L SNDX-275 0.2μmol/L) SNDX 275 0 2μmol/L)

0.8 0.6

* P <= 0.001 0 001 *

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04 0.4 0.2 0 2 0

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P-Akt P Akt Akt P MAPK P-MAPK MAPK β β-actin Fig. 4. The combinations of trastuzumab and SNDX-275 significantly inhibited cell proliferation of trastuzumab-resistant breast cancer cells associated with the inactivation of erbB2/erbB3 and downstream signaling. A, BT474-HR20 cells were plated onto 96-well plates and incubated at 37 °C with 5% CO2. After 24 h, the culture medium was replaced with 0.1 ml fresh medium containing 0.5% FBS or the same medium containing either trastuzumab (137 nmol/L), or the indicated concentrations (0.2, 0.5, 1.0 lmol/L) of SNDX-275 alone, or in combination of trastuzumab (137 nmol/L) and SNDX-275 for another 72 h incubation. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Bars, SD. Data shows a representative of at least three independent experiments. B, BT474-HR20 cells were treated with either trastuzumab, or SNDX-275 alone, or combinations of trastuzumab and SNDX-275 for 2 or 4 days. Cells were collected and subjected to western blot analyses with specific antibodies directed against erbB2, P-erbB2, erbB3, P-erbB3, P-Akt, Akt, P-MAPK, MAPK, or b-actin.

Fig. 5. Trastuzumab significantly accelerates SNDX-275-induced apoptosis in trastuzumab-resistant breast cancer cells. BT474-HR20 cells were treated with either trastuzumab, or SNDX-275 alone, or their combinations for 2 or 4 days. Cells were collected and subjected to western blot analyses with specific antibodies directed against PARP, Caspase-3, or bactin (A), or apoptotic-ELISA (B). Bars, SD. whereas it clearly reduced the expression levels of erbB3 in BT474HR20 cells (Fig. 6A). Treatment with SNDX-275 for 2 or 4 days did not significantly alter the interactions between IGF-1R and erbB2. However, the erbB3/IGF-1R associations were gradually decreased upon SNDX-275 treatment for 2 days. When BT474-HR20 cells were treated for 4 days, SNDX-275 exhibited an even profound reduction on the interactions between erbB3 and IGF-1R (Fig. 6B). These observations correlated with SNDX-275’s inhibitory effects on the expression levels of erbB3 protein (Fig. 6A). We then performed immunoprecipitation assays with an antierbB3 antibody followed by western blot analysis with an anti-erbB2 antibody. Treatment with SNDX-275 for 2 days dramatically decreased the interactions of erbB2 and erbB3, and furthermore the erbB2/erbB3 associations were undetectable when the cells were treated with SNDX275 for 4 days (Fig. 6C). Taken together, our results suggest that SNDX275 overcomes trastuzumab resistance mainly through downregulation of erbB3 levels and disruption of the interactions between erbB2 and erbB3 receptors in BT474-HR20 cells.

3.4. SNDX-275 disrupts the interactions between erbB2 and erbB3 receptors in trastuzumab-resistant breast cancer cells Our previously published data revealed that the three receptor tyrosine kinases (RTKs), erbB2, erbB3, and IGF-1R, formed a heterotrimeric complex to induce trastuzumab resistance [28]. Thus we tested whether SNDX-275 blocked the interactions between IGF-1R and erbB2, erbB3 and IGF-1R, or erbB2 and erbB3 in order to overcome trastuzumab resistance in BT474-HR20 cells. Upon treatment with SNDX-275 at 0.2 lmol/L or 0.5 lmol/L for 2 or 4 days, BT474-HR20 cells were collected and subjected to western blot and co-immunoprecipitation analyses to examine the expression levels of the three RTKs and their associations, respectively. SNDX-275 had little or no effect on expression of erbB2 and IGF-1R,

4. Discussion As a selective class I HDACi, SNDX-275 is a relatively safe drug and currently in phase I and/or II clinical trials for a variety of human cancers, including ER + breast cancers2. SNDX-275 is an orally administered drug with little toxicity to normal cells [30]. Its clinical activity has been tested in patients with advanced solid tumors or lymphoma [31–33]. Studies have shown that SNDX-275 is well

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0.5 SNDX-275 (μmol/L) erbB2 erbB3

Fig. 6. SNDX-275 mainly disrupts the interactions of erbB2/erbB3 receptors in trastuzumab-resistant breast cancer cells. BT474-HR20 cells were untreated or treated with the indicated concentrations of SNDX-275 for 2 or 4 days. Cells were collected and subjected to western blot analyses with specific antibodies directed against erbB2, erbB3, IGF-1R, or b-actin (A). The same cell lysates were also subjected to immunoprecipitation with an anti-IGF-1R antibody, and then followed by western blot analyses with specific antibodies directed against erbB2, erbB3, or IGF-1R (B). The same cell lysates were also subjected to immunoprecipitation with an anti-erbB3 antibody, and then followed by western blot analyses with specific antibodies directed against erbB2 or erbB3 (C).

tolerated at doses up to 6 mg/m2 administered weekly [31,32]. The maximum-tolerated dose (MTD) of SNDX275 to cancer patients is 10 mg/m2 [33]. While administered at 8 mg/m2, the peak plasma concentration of SNDX-275 is about 146.9 ng/mL or 0.39 lmol/L [32]. Since our recent data suggest that SNDX-275 preferably inhibits cell proliferation and induces apoptosis in erbB2-overexpressing breast cancer cells in a dose-dependent manner [23], we on purposely used the clinically achievable concentrations of SNDX-275 to explore its combinational potential with trastuzumab against trastuzumab-sensitive and -resistant breast cancer cells. Two treatment regimens were performed to mimic the clinical conditions in the present study: 1) Trastuzumab (137 nmol/L) and/or SNDX-275 (0.5 lmol/L) for 2 days; and 2) Trastuzumab (68.5 nmol/L) and/or SNDX-275 (0.2 lmol/L) for 4 days. SNDX-275 in combination with trastuzumab not only enhanced trastuzumab efficacy against erbB2-overexpressing breast cancer cells, but also significantly re-sensitized those resistant cells to trastuzumab-induced growth inhibition (Figs. 1 and 4, S1 and S2). The major combinational

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effects of trastuzumab and SNDX-275 were to inhibit cell proliferation and induce caspase-dependent apoptosis via inactivation of erbB2/erbB3 receptor and their downstream signaling Akt (Figs. 2–5). To the best of our knowledge, this is the first report showing that SNDX-275 is able to enhance the efficacy of trastuzumab against both trastuzumab-sensitive and -resistant erbB2-overexpressing breast cancer cells. Our studies strongly suggest that the novel strategy to combine SNDX-275 and trastuzumab is a promising approach to treat those breast cancer patients with erbB2-overexpressing tumors. We believe that the toxicities might be very low and well tolerated when the dosage of SNDX-275 is reduced to 0.2 lmol/L and trastuzumab to 68.5 nmol/L. Nonetheless, a clinical trial is needed to confirm the safety of this novel strategy. One of our important findings was that SNDX-275 significantly blocked the interactions between erbB2 and erbB3 receptors in trastuzumab-resistant breast cancer cells. Our previously published data showed that the erbB2/erbB3 interactions in trastuzumab-resistant breast cancer cells were much higher than that in the parental trastuzumab-sensitive cells [28]. It has been well-documented that the erbB2/erbB3 heterodimer functions as one oncogenic unit to promote breast cancer cell proliferation [34]. It generally signals through MEK/MAPK and/or PI-3K/Akt signaling pathways to modulate many important downstream regulators that play a pivotal role in maintaining malignant phenotypes, including cell proliferation, resistance, angiogenesis, invasion, and survival [35,36]. Disruption of the erbB2/erbB3 associations by SNDX-275 resulted in a dramatic inhibition on the PI-3K/Akt signaling, the major determinant of trastuzumab resistance in breast cancer [27]. Thus, it is conceivable to hypothesize that inactivation of PI-3K/Akt signaling may be one of the mechanisms through which SNDX-275 overcomes trastuzumab resistance. At this moment, however, it is not known whether SNDX-275 may also hold potential to abrogate therapeutic resistance to lapatinib, another erbB2-targeted therapy approved by FDA to treat metastatic breast cancer (MBC) that has progressed after trastuzumab-based therapy [37]. Unfortunately, the efficacy of this erbB2 tyrosine kinase inhibitor is also compromised by both primary and acquired resistance [38,39]. Due to lack of preclinical models, the molecular mechanisms of lapatinib resistance remain unclear. Increased evidence reveals that lapatinib and trastuzumab do not share common mechanisms of therapeutic resistance, as lapatinib has activity in trastuzumab-resistant breast cancers [40–43]. Although previous studies suggest that lapatinib exerts its anti-tumor activity in a PTEN independent manner [44], a recent report indicates that loss of PTEN and the resulting activation of PI-3K/Akt signaling lead to lapatinib resistance [45]. While a synergistic effect of lapatinib and SNDX-275 was observed in BT474 and inflammatory breast cancer cells [46], the combinations of these two agents have not been tested in trastuzumab-resistant breast cancer cells. Thus, it will be very interesting and in clinical relevance to study if SNDX-275 and lapatinib may exhibit synergistic or additive effects on growth inhibition and/or apoptosis in BT474-HR20 and SKBR3pool2 cells.

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In addition to inducing trastuzumab resistance, activation of the erbB2/erbB3/PI-3K/Akt signaling pathway also plays a critical role in erbB2-mediated resistance to antiestrogen tamoxifen [47] and chemotherapeutic drug paclitaxel [48] in breast cancer treatment. we have recently reported that specific knockdown of erbB3 expression abrogates erbB2-mediated tamoxifen resistance [49], and enhances paclitaxel-induced apoptosis in erbB2overexpressing breast cancer cells [29]. The ability of SNDX-275 to reduce erbB3 levels and inactivate Akt in erbB2-overexpressing breast cancer cells support the hypothesis that SNDX-275 may abrogate or attenuate erbB2-mediated therapeutic resistance to tamoxifen and paclitaxel. We are currently testing this hypothesis in the laboratory. To date, the molecular mechanisms of SNDX275-induced downregulation of erbB3 remain unclear. Our recent studies suggest that SNDX-275 reduces erbB3 levels via transcription-independent mechanisms. Further investigation on such molecular mechanisms may provide new avenues to identify novel strategies targeting erbB3 receptor and its downstream signaling PI-3K/Akt pathway for breast cancer treatment. In summary, we have shown that SNDX-275 significantly enhances trastuzumab-induced growth inhibition and apoptosis in erbB2-overexpressing breast cancer cells, and possesses therapeutic potential to overcome trastuzumab resistance. SNDX-275’s major anti-cancer activity is to downregulate erbB3 protein levels, disrupt erbB2/ erbB3 dimerization, and subsequently inactivate the PI-3K/Akt signaling. Our studies may provide a novel combinational strategy to treat erbB2-overexpressing breast cancer patients, especially for those whose tumors have already developed resistant to trastuzumab. Conflict of interests None declared. Acknowledgements The authors are grateful to Dr. Peter Ordentlich (Syndax Pharmaceuticals, Inc., Waltham, MA) for providing SNDX275, to Dr. Francisco Esteva (MD Anderson Cancer Center) for providing SKBR3-pool2 cells. This work was supported in part by a Susan G. Komen for the Cure Research Grant (BCTR0707449) and AWD-102888 from the Cancer League of Colorado (to BL). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2011. 03.019. References [1] D.J. Slamon, G.M. Clark, S.G. Wong, W.J. Levin, A. Ullrich, W.L. McGuire, Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene, Science 235 (1987) 177–182.

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