Trastuzumab-resistant Breast Cancer Cells Research Articles

ADAM10 mediates trastuzumab resistance and is correlated with survival in HER2 positive breast cancer.

Trastuzumab prolongs survival in HER2 positive breast cancer patients. However, resistance remains a challenge. We have previously shown that ADAM17 plays a key role in maintaining HER2 phosphorylation during trastuzumab treatment. Beside ADAM17, ADAM10 is the other well characterized ADAM protease responsible for HER ligand shedding. Therefore, we studied the role of ADAM10 in relation to trastuzumab treatment and resistance in HER2 positive breast cancer. ADAM10 expression was assessed in HER2 positive breast cancer cell lines and xenograft mice treated with trastuzumab. Trastuzumab treatment increased ADAM10 levels in HER2 positive breast cancer cells (p ≤ 0.001 in BT474; p ≤ 0.01 in SKBR3) and in vivo (p ≤ 0.0001) compared to control, correlating with a decrease in PKB phosphorylation. ADAM10 inhibition or knockdown enhanced trastuzumab response in naïve and trastuzumab resistant breast cancer cells. Trastuzumab monotherapy upregulated ADAM10 (p ≤ 0.05); and higher pre-treatment ADAM10 levels correlated with decreased clinical response (p ≤ 0.05) at day 21 in HER2 positive breast cancer patients undergoing a trastuzumab treatment window study. Higher ADAM10 levels correlated with poorer relapse-free survival (p ≤ 0.01) in a cohort of HER2 positive breast cancer patients. Our studies implicate a role of ADAM10 in acquired resistance to trastuzumab and establish ADAM10 as a therapeutic target and a potential biomarker for HER2 positive breast cancer patients.

MiR-200c suppresses TGF-β signaling and counteracts trastuzumab resistance and metastasis by targeting ZNF217 and ZEB1 in breast cancer

Resistance to trastuzumab and concomitantly distal metastasis are leading causes of mortality in HER2-positive breast cancers, the molecular basis of which remains largely unknown. Here, we generated trastuzumab-resistant breast cancer cells with increased tumorigenicity and invasiveness compared with parental cells, and observed robust epithelial-mesenchymal transition (EMT) and consistently elevated TGF-β signaling in these cells. MiR-200c, which was the most significantly downregulated miRNA in trastuzumab-resistant cells, restored trastuzumab sensitivity and suppressed invasion of breast cancer cells by concurrently targeting ZNF217, a transcriptional activator of TGF-β, and ZEB1, a known mediator of TGF-β signaling. Given the reported backward inhibition of miR-200c by ZEB1, ZNF217 also exerts a feedback suppression of miR-200c via TGF-β/ZEB1 signaling. Restoration of miR-200c, silencing of ZEB1 or ZNF217 or blockade of TGF-β signaling increased trastuzumab sensitivity and suppressed invasiveness of breast cancer cells. Therefore, our study unraveled nested regulatory circuits of miR-200c/ZEB1 and miR-200c/ZNF217/TGF-β/ZEB1 in synergistically promoting trastuzumab resistance and metastasis of breast cancer cells. These findings provide novel insights into the common role of EMT and related molecular machinery in mediating the malignant phenotypes of breast cancers.

Abstract P5-11-03: Deciphering the role of HER3-DJ-1 interaction in breast cancer

Abstract Advanced breast cancer is notorious for its aggressive nature and poor prognosis. An ablated regulation in the EGFR family receptor signaling has been linked to the development of numerous tumor types, including breast cancer. HER3 is a unique member of the EGFR family that lacks intrinsic tyrosine kinase activity. However, HER3 can initiate signaling when dimerized with other family members such as HER2. HER3 overexpression is correlated with poor survival in breast cancer patients. HER3 also plays an important role in drug resistance mechanism such as in trastuzumab resistant breast cancer cell lines where it dimerize with EGFR to initiate signaling via the PI3K/Akt pathway. The identification of interaction partners of HER3's C-tail is critical to understand the signaling mechanism underpinning HER3's role in drug resistance and in progression of breast cancer. Our laboratory has identified a panel of novel HER3-interacting proteins by a combined immunoprecipitation and mass-spectrometry approach. One of the novel HER3 interacting proteins is DJ-1 which is a chaperone protein that plays an important role in the development of many cancers, including breast cancer. DJ-1 knockdown in high HER3 expressing breast cancer cell lines such as MCF-7 and MDA- MB-453 resulted in the reduction in HER3 levels as well as proteins downstream of the HER3 signaling pathway such as MAPK. We have also demonstrated that DJ-1 knockdown led to decrease in cancer cell proliferation. Studies are now in progress to determine the mechanism of regulation of HER3 by DJ-1 and the impact of DJ-1 KD in in vivo breast cancer xenograft models. These studies will help us to decipher key molecules which regulate HER3 mediated signaling and their role in breast cancer development and progression. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-03.

Abstract P5-08-03: In vitro resistance to ERBB2-targeted therapies does not bestow cross-resistance to antibody dependent cellular cytotoxicity

Abstract Background: The advent of ERBB2-directed therapies, including trastuzumab and lapatinib, has changed the clinical management of ERBB2+ breast cancer and improved patient outcomes. However, both de novo and acquired resistance to ERBB2-targeting agents remain significant clinical problems; thus, it is important to understand the mechanisms that tumor cells use to resist and/ or escape ERBB2-directed therapeutic agents. A growing body of evidence supports the role of immune effector cells in trastuzumab-induced tumor cell lysis in vivo, which is hypothesized to occur through antibody dependent cellular cytotoxicity (ADCC) mediated by natural killer cells bearing FC gamma receptors. Existing in vitro models of trastuzumab and lapatinib resistance are limited in that they are most often generated by incubating tumor cells with drug alone, outside the context of the host immune system. Consequently, the impact of in vitro generated drug resistance on ADCC susceptibility has not been defined. In this study, we tested existing trastuzumab and lapatinib resistant breast cancer cell lines for ADCC sensitivity. Materials and Methods: The ERBB2 amplified breast cancer cell lines BT474 and SKBR3, which are sensitive to trastuzumab-mediated ADCC, were selected for trastuzumab and lapatinib resistance. Drug resistant cells were generated by serial culture in the presence of escalating doses of trastuzumab and/or lapatinib until complete tolerance was acquired at clinically achievable concentrations (100μg/ml trastuzumab and 2.6μM lapatinib). Drug sensitivity was assessed by Alamar Blue assay. Cell resistance to trastuzumab-mediated ADCC was measured by Calcein-AM release assay, using 1μg/ml antibody concentration and four different effector target ratios (20:1, 10:1, 5:1, and 1:1), with a minimum of five replicates. Human peripheral blood lymphocytes freshly isolated from buffy coat were used as effector cells. Results and Discussion: Neither BT474 nor SKBR3 cells with acquired resistance to single agent trastuzumab (HerR), lapatinib (LapR), or both in combination (DualR) demonstrated cross-resistance to effector cell-mediated ADCC at any of the E:T ratios tested. At the highest E:T ratio tested (20:1), trastuzumab-induced ADCC of control BT474 cells resulted in 61.92% cell lysis (95% CI = 3.97), compared to 64.97% lysis for BT-HerR cells (95% CI = 2.87, Student's t-test p > 0.1). Similarly, BT-LapR and DualR cell lysis was 58.13% and 63.66%, respectively (95% CI = 2.62 and 6.57, Student's t-test p > 0.1). Additionally, trastuzumab-induced ADCC of control SKBR3 cells resulted in 89.98% cell lysis (95% CI = 4.29), compared to 93.53% lysis for SK-HerR cells (95% CI = 0.81, Student's t-test p > 0.1). SK-LapR and DualR cell lysis was 94.81% and 92.09%, respectively (95% CI = 0.6 and 2.66, Student's t-test p > 0.1). Our results indicate the importance of incorporating immune effector cells to in vitro models of trastuzumab resistance. Accordingly, we have begun modeling ADCC resistance in vitro to further investigate the relationship between targeted antibody therapies, effector cell-mediated cytolysis, and mechanisms of tumor immune escape. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-03.

Abstract P4-12-14: Metal chelating polymer-conjugated trastuzumab labeled to high specific activity with indium-111 is cytotoxic to trastuzumab-sensitive and resistant breast cancer cells through the emission of auger electrons

Abstract Purpose: Auger electrons are very low energy electrons emitted by Indium-111(111In) that cause lethal DNA damage when emitted in close to the cell nucleus. Our laboratory reported previously that trastuzumab modified with nuclear localizing sequence (NLS) peptides and labeled with 111In(111In-NLS-DTPA-trastuzumab) was internalized and transported to the nucleus of HER2-overexpressing breast cancer (BC) cells, reducing their clonogenic survival (CS) to ≤10%. However, a limitation was that at the specific activity (SA) achieved (0.24 MBq/μg) for 111In-DTPA-trastuzumab without or with NLS, only 1 in 50 molecules was radiolabeled. This reduced the potency for cells with low or intermediate HER2 density. Our purpose was to dramatically increase the SA of 111In-labeled trastuzumab by conjugation to a metal chelating polymer (MCP) that displays multiple DTPA chelators for 111In and compare its potency to 111In-NLS-trastuzumab on BC cells with a wide range of HER2 density including cells resistant to trastuzumab. Methods: An MCP with a polyglutamide backbone, 29 DTPA groups, and a hydrazide reactive end-group was site-specifically linked to periodate-oxidized carbohydrates on the Fc domain of trastuzumab. HER2 immunoreactivity, internalization and nuclear importation were evaluated by cell binding and fractionation studies using HER2-overexpressing SK-Br-3 human BC cells. The SA achievable was assessed by radiolabeling 1, 5, 50 and 100 μg of the immunoconjugates with 9.25 MBq of 111In. The effect of high SA (5.5 MBq/μg) 111In-MCP-trastuzumab to on the CS of human BC cells with a wide range of HER2 density (5×105-1.3×106 HER2 receptors/cell) that were sensitive or resistant to trastuzumab was compared to low SA (0.37 MBq/μg) 111In-labeled trastuzumab conjugates. Studies are also in progress to assess DNA double-strand breaks caused by the Auger electrons emitted by 111In using the γH2AX assay. Results: MCP-trastuzumab bound specifically to HER2 with high affinity (KD = 10.7±1.5nM) and was internalized and imported into the nucleus of HER2-positive BC cells. The smallest mass that could be labeled (labeling efficiency>80%) with 111In for MCP-trastuzumab was 1 μg (SA = 8.9 MBq/μg) compared to 50 μg for DTPA-trastuzumab (SA = 0.08 MBq/μg). High SA 111In-MCP-trastuzumab reduced the CS of SK-Br-3 cells with high HER2 density to 1.8±1.3% and the CS of TrR1 trastuzumab-resistant BC cells with intermediate HER2 density to 17.1±1.6%. These represent a 4-5 and 5-6 fold greater potency compared to low SA 111In-trastuzumab conjugates on cells with high or intermediate HER2 expression, respectively. Conclusion: 111In-MCP-trastuzumab was labeled to 37-fold higher SA than 111In-DTPA-trastuzumab. High SA increased its cytotoxicity potency for HER2-positive BC cells that were sensitive or resistant to trastuzumab. Auger electron radioimmunotherapy is a promising strategy for HER2-positive BC which may extend to trastuzumab-resistant tumours. Supported by a grant from the Canadian Breast Cancer Research Alliance with funds from the Canadian Cancer Society. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-14.

Identification of quinones as HER2 inhibitors for the treatment of trastuzumab resistant breast cancer

HER2 overexpression is associated with aggressive breast cancer with high recurrence rate and poor patient prognosis. Treatment of HER2 overexpressing patients with the HER2 targeting therapy trastuzumab results in acquired resistance within a year. The HER2/EGFR dual kinase inhibitor lapatinib was shown to inhibit some trastuzumab resistant breast cancer cell lines and is currently in clinical trials. Our group has found two new quinone compounds that show excellent inhibition of breast tumor cells expressing HER2 or the trastuzumab resistant HER2 oncogenic isoform, HER2Δ16. Compound 4 ((1R,2S,3S)-1,2,3,5,8-pentahydroxy-1,2,3,4-tetrahydroanthracene-9,10-dione) and compound 5 (5,8-dihydroxy-2,3-bis(hydroxymethyl)naphthalene-1,4-dione) showed sub-micromolar inhibition potency against these cell lines. These compounds also inhibit auto-phosphorylation of the Y1248 and Y1068 residues of HER2 and EGFR, respectively.

The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

BackgroundElevated expression of erbB3 receptor has been reported to induce resistance to therapeutic agents, including trastuzumab in erbB2-overexpressing breast cancer. Our recent studies indicate that erbB3 interacts with both erbB2 and IGF-1 receptor to form a heterotrimeric complex in trastuzumab-resistant breast cancer cells. Herein, we investigate the antitumor activity of MM-121/SAR256212, a fully human anti-erbB3 antibody (Ab), against two erbB2-overexpressing breast cancer cell lines resistant to trastuzumab.MethodsMTS-based proliferation assays were used to determine cell viability upon treatment of trastuzumab and/or MM-121/SAR256212. Cell cycle progression was examined by flow cytometric analysis. Western blot analyses were performed to determine the expression and activation of proteins. Tumor xenografts were established by inoculation of the trastuzumab-resistant BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with trastuzumab and/or MM-121/SAR256212 via i.p injection to determine the Abs’ antitumor activity. Immunohistochemical analyses were carried out to study the Abs’ inhibitory effects on tumor cell proliferation and induction of apoptosis in vivo.ResultsMM-121 significantly enhanced trastuzumab-induced growth inhibition in two sensitive and two resistant breast cancer cell lines. MM-121 in combination with trastuzumab resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and Akt (P-Akt) in the in vitro studies. MM-121 combined with trastuzumab did not induce apoptosis in the trastuzumab-resistant cell lines under our cell culture condition, rather induced cell cycle G1 arrest mainly associated with the upregulation of p27kip1. Interestingly, in the tumor xenograft model established from the trastuzumab-resistant cells, MM-121 in combination with trastuzumab as compared to either agent alone dramatically inhibited tumor growth correlated with a significant reduction of Ki67 staining and increase of cleaved caspase-3 in the tumor tissues.ConclusionsThe combination of MM-121 and trastuzumab not only inhibits erbB2-overexpressing breast cancer cell proliferation, but also promotes the otherwise trastuzumab-resistant cells undergoing apoptosis in an in vivo xenografts model. Thus, MM-121 exhibits potent antitumor activity when combined with trastuzumab under the studied conditions. Our data suggest that further studies regarding the suitability of MM-121 for treatment of breast cancer patients whose tumors overexpress erbB2 and become resistant to trastuzumab may be warranted.

Bispecific Antibody to ErbB2 Overcomes Trastuzumab Resistance through Comprehensive Blockade of ErbB2 Heterodimerization

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. However, resistance to trastuzumab is common. Heterodimerization between ErbB2 and other ErbBs may redundantly trigger cell proliferation signals and confer trastuzumab resistance. Here, we developed a bispecific anti-ErbB2 antibody using trastuzumab and pertuzumab, another ErbB2-specific humanized antibody that binds to a distinct epitope from trastuzumab. This bispecific antibody, denoted as TPL, retained the full binding activities of both parental antibodies and exhibited pharmacokinetic properties similar to those of a conventional immunoglobulin G molecule. Unexpectedly, TPL showed superior ErbB2 heterodimerization-blocking activity over the combination of both parental monoclonal antibodies, possibly through steric hindrance and/or inducing ErbB2 conformational change. Further data indicated that TPL potently abrogated ErbB2 signaling in trastuzumab-resistant breast cancer cell lines. In addition, we showed that TPL was far more effective than trastuzumab plus pertuzumab in inhibiting the growth of trastuzumab-resistant breast cancer cell lines, both in vitro and in vivo. Importantly, TPL treatment eradicated established trastuzumab-resistant tumors in tumor-bearing nude mice. Our results suggest that trastuzumab-resistant breast tumors remain dependent on ErbB2 signaling and that comprehensive blockade of ErbB2 heterodimerization may be an effective therapeutic avenue. The unique potential of TPL to overcome trastuzumab resistance warrants its consideration as a promising treatment in the clinic.

Abstract A070: The erbB3 blocking antibody MM-121 resensitizes trastuzumab-resistant breast cancer cells to trastuzumab-mediated antitumor activity in vitro and in vivo

Abstract Background: Amplification and/or overexpression of erbB2 occur in approximately 25% of invasive breast cancer and are significantly associated with a worse prognosis for breast cancer patients. The erbB3 receptor plays a critical role in erbB2-overexpressing breast cancer, and its elevated expression induces resistance to therapeutic agents, including trastuzumab. Our recent studies indicate that erbB3 interacts with both erbB2 and IGF-1 receptor to form a heterotrimeric complex in trastuzumab-resistant breast cancer cells. Herein, we investigate the antitumor activity of MM-121, a humanized anti-erbB3 antibody, against erbB2-overexpressing breast cancer cells resistant to trastuzumab in vitro and in vivo. Material and Methods: Cell Titer AQueous Non-Radioactive Cell Proliferation Assay (MTS) to determine cell proliferation. Flow Cytometry analyses to examine cell cycle distribution. Western blot analysis to evaluate protein expression and activation upon treatment. Tumor xenograft model to explore the combinatorial effects of MM-121 and trastuzumab on tumor growth of trastuzumab-resistant breast cancer cells in vivo. Immunohistochemistry assays to assess the treatment effects on expression of erbB3, erbB2, Ki-67, and cleaved capase-3 in vivo. Results: Cell proliferation assays showed that MM-121 significantly enhanced trastuzumab-induced growth inhibition in both sensitive and resistant breast cancer cells. MM-121 in combination with trastuzumab mainly resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and the downstream signaling P-Akt. MM-121 combined with trastuzumab did not lead to apoptosis in trastuzumab-resistant cells in in vitro cell culture condition, rather induced cell cycle G1 arrest which was associated with a slight decrease of E2F-1 and upregulation of p27kip1. Interestingly, in the mouse tumor xenografts model established from trastuzumab-resistant cells, MM-121 in combination with trastuzumab as compared to either agent alone significantly inhibited tumor growth correlated with a significant reduction of Ki-67 staining and increase of cleaved caspase-3 in the tumor tissues. These data suggest that the combination of MM-121 and trastuzumab not only inhibits tumor cell proliferation, but also promotes the resistant cells undergoing apoptosis in vivo. Conclusions: We demonstrate that MM-121 exhibits potent antitumor activity when combined with trastuzumab in vitro and in vivo. MM-121 may be added into the treatment regimens for breast cancer patients whose tumors overexpress erbB2 and become resistant to trastuzumab. Citation Format: Jingcao Huang, Hui Lyu, Youngseok Lee, Bolin Liu. The erbB3 blocking antibody MM-121 resensitizes trastuzumab-resistant breast cancer cells to trastuzumab-mediated antitumor activity in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A070.

Abstract B042: Differential effects of erbB3 and IGF-1R signaling on lapatinib resistance acquired by the erbB2-overexpressing breast cancer cells refractory to trastuzumab

Abstract Introduction: Resistance to erbB2-targeted therapy frequently occurs in the treatment of erbB2-overexpression breast cancer. We previously showed that the heterotrimerization of erbB2, erbB3, and IGF-1 receptor (IGF-1R) resulted in trastuzumab resistance. Enhanced activation of the downstream signaling, such as PI3K/Akt and Src, was related to the resistant phenotype. Specific knockdown of either erbB3 or IGF-1R significantly reversed the resistance, suggesting that both erbB3 and IGF-1R signaling contributed to trastuzumab resistance. However, it is not known whether the trastuzumab-resistant breast cancer cells are also refractory to lapatinib, another targeted therapeutic against erbB2. It remains unclear whether the erbB3 and IGF-1R signaling may differentially influence the lapatinib sensitivity. Material and Methods: Cell growth (MTS) assays were used to determine cell viability. Quantification of cells undergoing apoptosis was measured by a specific apoptotic ELISA. Western blot analyses were performed to assess the expression and activation of proteins. Lentiviral vector containing shRNA was used to specifically knockdown erbB3 or IGF-1R. Results: The acquired trastuzumab-resistant breast cancer cells (SKBR3-pool2 and BT474-HR20 derived from SKBR3 and BT474 cells, respectively) also showed cross-refractoriness to lapatinib. The IC50s of lapatinib in SKBR3 and SKBR3-pool2 cells were 0.043±0.003 μmol/L and 0.15±0.003 μmol/L, respectively. The IC50s of lapatinib in BT474 and BT474-HR20 cells were 0.054±0.009 μmol/L and 0.12±0.006 μmol/L (p<0.05), respectively. These data suggest heterotrimerization of the erbB2, erbB3, and IGF-1R may also lead to lapatinib resistance. To further elucidate the role of erbB3 and IGF-1R in the lapatinib resistance, the lentiviral vectors containing specific shRNA were used. Specific knockdown of erbB3 in both SKBR3-pool2 and BT474-HR20 cells significantly overcame the resistant phenotype, and dramatically re-sensitized the cells to lapatinib-induced apoptosis. Meanwhile, inactivation of the downstream PI3K/Akt signaling was clearly detected. In contrast, specific knockdown of IGF-1R did not alter the cells' refractoriness to lapatinib treatment. While the levels of phosphorylated Src (P-Src) were reduced upon IGF-1R knockdown, the levels of P-Akt, however, remained unchanged. Conclusions: The erbB2-overexpressing breast cancer cells resistant to trastuzumab also show refractory to Lapatinib, which may be attributed to the heterotrimerization of erbB2, erbB3, and IGF-1R as well. Our data indicate that the erbB3 receptor plays an important role in both trastuzumab and lapatinib resistances mainly through activation of the PI3K/Akt signaling. However, the IGF-1R, via activation of the Src kinase, only leads to trastuzumab resistance without affecting the cells' sensitivity to lapatinib. To the best of our knowledge, this is the first report experimentally demonstrating that the erbB3 and IGF-1R signaling differentially regulates lapatinib sensitivity in trastuzumab-resistant breast cancer cells. These findings may facilitate the development of novel therapeutic regimens for breast cancer treatment. Citation Format: Hui Lyu, Jingcao Huang, Youngseok Lee, Bolin Liu. Differential effects of erbB3 and IGF-1R signaling on lapatinib resistance acquired by the erbB2-overexpressing breast cancer cells refractory to trastuzumab. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B042.

Reconstruction of Cellular Signal Transduction Networks Using Perturbation Assays and Linear Programming

Perturbation experiments for example using RNA interference (RNAi) offer an attractive way to elucidate gene function in a high throughput fashion. The placement of hit genes in their functional context and the inference of underlying networks from such data, however, are challenging tasks. One of the problems in network inference is the exponential number of possible network topologies for a given number of genes. Here, we introduce a novel mathematical approach to address this question. We formulate network inference as a linear optimization problem, which can be solved efficiently even for large-scale systems. We use simulated data to evaluate our approach, and show improved performance in particular on larger networks over state-of-the art methods. We achieve increased sensitivity and specificity, as well as a significant reduction in computing time. Furthermore, we show superior performance on noisy data. We then apply our approach to study the intracellular signaling of human primary nave CD4+ T-cells, as well as ErbB signaling in trastuzumab resistant breast cancer cells. In both cases, our approach recovers known interactions and points to additional relevant processes. In ErbB signaling, our results predict an important role of negative and positive feedback in controlling the cell cycle progression.

Autocrine/paracrine mechanism of interleukin-17B receptor promotes breast tumorigenesis through NF-κB-mediated antiapoptotic pathway

Gain of function of membrane receptor was a good strategy exploited by cancer cells to benefit own growth and survival. Overexpression of HER2 has been found to serve as a target for developing trastuzumab to treat 20-25% of breast cancer. However, little or none of the other membrane receptor was found to be useful as a potential target for breast cancer treatment since then. Here, we showed that amplified signaling of interleukin-17 receptor B (IL-17RB) and its ligand IL-17B promoted tumorigenicity in breast cancer cells and impeded acinus formation in immortalized normal mammary epithelial cells. External signal transmitted through IL-17RB activated nuclear factor-κB to upregulate antiapoptotic factor Bcl-2 and induced etoposide resistance. Elevated expression of IL-17RB had a stronger correlation with poor prognosis than HER2 in breast cancer patients. Interestingly, breast cancer patients with high expression of IL-17RB and HER2 had the shortest survival rate. Depletion of IL-17RB in trastuzumab-resistant breast cancer cells significantly reduced their tumorigenic activity, suggesting that IL-17RB and HER2 have an independent role in breast carcinogenesis. Furthermore, treatment with antibodies specifically against IL-17RB or IL-17B effectively attenuated tumorigenicity of breast cancer cells. These results suggest that the amplified IL-17RB/IL-17B signaling pathways may serve as a therapeutic target for developing treatment to manage IL-17RB-associated breast cancer.

Trastuzumab-Resistant Cells Rely on a HER2-PI3K-FoxO-Survivin Axis and Are Sensitive to PI3K Inhibitors

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.

Abstract P4-08-06: Notch-dependent Regulation of Novel Genes Associated with Trastuzumab Resistance

Abstract Background: We have shown previously that Notch-1 is critical for the development and maintenance of trasuzumab or lapatinib resistance in HER2 positive breast cancer cell lines. Here we sought to identify the critical genes that are regulated by Notch to promote anti-HER2 targeted resistance. Methods: We measured expression of 84 known breast cancer-associated transcripts using a real-time PCR array in both trastuzumab sensitive and resistant BT474 cell lines. A 4 fold increase or decrease in transcripts levels were measured to be statisitically significant. To confirm the change in levels, we designed primers to each gene of interest and repeated the PCR at least three independent times. Furthermore, we specifically asked whether Notch-1 or it's transcriptional mediator, CBF-1, directly regulated any of the identified genes in HER2 positive breast cancer cells using a genetic knocked down approach. An ANOVA for multiple comparisons was used to compute statistical significance. Results: We identified more than 4 fold increase in APC, ABCG2, ABCB1, Gata-3, Bcl-2, Id2, and HRG-2 transcripts in trastuzumab resistant versus sensitive cells. Conversley, Gli1 was downregulated in resistant versus sensitive cells. Interestingly, siRNA directed against Notch-1 or CBF-1 decreased ABCG2 and Gata-3, but conversely increased ABCB1 and HRG-2 in resistant cells. Conclusions: Our findings demonstrate APC, ABCG2, ABCB1, Gata-3, Bcl-2, Id2, and HRG-2 transcripts are signficantly increased in trastuzumab resistant breast cancer cells compared to sensitive cells. More importantly, Notch-1 or it's direct transcriptional activator CBF-1 may contribute to traztuzumab resistance by directly regulating critical genes that contribute to multidrug resistance: ABCB1 and ABCG2 and/or genes associated with stem cell survival: Gata-3 and HRG-2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-06.

Abstract B31: Expression of Wnt3 activates Wnt/β-catenin pathway and promotes EMT-like phenotype in trastuzumab resistant HER2-overexpressing breast cancer cells.

Abstract To understand the mechanisms leading to trastuzumab resistance in HER2-overexpressing breast tumors we created trastuzumab insensitive cell lines (SKBR3/100-8 and BT474/100-2). The cell lines maintain HER2 receptor overexpression, and show increase in EGFR. Upon trastuzumab treatment, SKBR3/100-8 and BT474/100-2 cell lines displayed increased growth rate and invasiveness. The trastuzumab resistance in SKBR3/100-8 and BT474/100-2 was accompanied with activation of the Wnt/β-catenin signaling pathway. Further investigation found that Wnt3 overexpression played a key role toward the development of trastuzumab resistance. The expression of Wnt3 in trastuzumab resistant cells increased nuclear expression of β-catenin and transactivated expression of EGFR. The increased Wnt3 in the trastuzumab resistant cells also promoted a parental EMT-like transition (epithelial to mesenchymal transition), increased N-cadherin, Twist, SLUG and decreased E-cadherin. Knockdown Wnt3 by siRNA restored cytoplasmic expression of β-catenin, and decreased EGFR expression in trastuzumab resistant cells. Furthermore the EMT markers were decreased, E-cadherin was increased and the cell invasiveness was inhibited in response to the Wnt3 down-regulation. Conversely, SKBR3 cells which had been stably transfected with full-length Wnt3 exhibited EMT-like transition. The Wnt3 transfectants, SKBR3/Wnt3-7 and SKBR3/Wnt3-9, showed significant decrease in E-cadherin and increase in N-cadherin, Twist and SLUG. The cells were less sensitive to trastuzumab compared to parental SKBR3 and vector transfected cells. In summary, our data suggests that Wnt3 overexpression activates Wnt/β-catenin signaling pathway that leads to transactivation of EGFR and promotes EMT-like transition. This could be an important mechanism leading to trastuzumab resistance in HER2 overexpressing breast cancer cells. Citation Format: Yanyuan Wu, Charles Ginther, Juri Kim, Nicole Mosher, Seyung Chung, Dennis Slamon, Jaydutt V. Vadgama. Expression of Wnt3 activates Wnt/β-catenin pathway and promotes EMT-like phenotype in trastuzumab resistant HER2-overexpressing breast cancer cells. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B31.

Abstract 4865: Pak1 links the Wnt/β-catenin pathway to ErbB2 signaling in breast cancer cells

Abstract Several studies suggest that Pak1 plays an important role in the genesis of breast cancer, but despite the similarity in their sequences, the contribution of the closely related enzyme Pak2 to this disease remains unclear. To establish the function of Paks in ErbB2 driven transformation, we used a three-dimensional tissue culture system of human mammary epithelial cells that stably co-express a chemically-activatable form of ErbB2 and doxycycline (DOX) inducible shRNAs against Pak1 or Pak2. We observed that silencing of Pak1 but not of Pak2 has a dramatic effect in cell proliferation and apoptosis. As expected, activation of ErbB2 induced growth factor-independent proliferation and disruption of 3D acinar-like structures via ERK and Akt signaling. In contrast, silencing of Pak1 compromised activation of ERK, resulting in reversion of the malignant phenotype and restoration of normal acinar architecture. Surprisingly, silencing of Pak2 also compromised ERK activation, but cannot restore normal morphology. Further, we found that ErbB2-amplified breast cancer cells expressing a DOX inducible shRNA against Pak1 formed significantly smaller tumors than cells expressing either GFP or a DOX inducible shRNA against Pak2 in SCID mice as the result of the inhibition of ERK. A phosphoproteomic analysis showed that several signaling pathways, e.g. Wnt and Mdm2-p53, are down regulated in our Pak1 deficient cell line. We found that the shPak1 cell line, but not the shPak2 deficient cell line has a dramatic reduction in total β-catenin levels, and that blocking β-catenin with chemical inhibitors restored normal acinar architecture. Finally, we found that targeting Pak and/or β-catenin in combination with trastuzumab sensitized trastuzumab-resistant breast cancer cells to trastuzumab, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance. 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 4865. doi:1538-7445.AM2012-4865

Abstract 1912: Jab1/Csn5 as a novel driver for therapeutic resistance in HER2-positive breast cancer

Abstract Her2-positive (Her2+) breast cancer has the second worst prognosis of the five subtypes of breast cancer. The most successful targeted therapy for Her2+ breast cancer to date is trastuzumab (Herceptin)-based. However, the median duration of response to trastuzumab is shorter than 1 year, and about 75% of patients who initially responsed, has experienced resistance to the therapy after a year. Thus, better treatment strategies for Her2+ subtype are urgently needed. We and other researchers previously demonstrated that c-Jun activation domain-binding protein 1 (Jab1) negatively regulates p27, mediates p27 nuclear-to-cytoplasmic export and degradation and contributes to the loss of p27 that is seen in >50% of breast tumors and that correlates with poor clinical outcome in breast cancer cells. Thus, implicates the potential role of Jab1 in interfering with trastuzumab. We identified Jab1/Csn5 as a novel oncogene. Jab1 is amplified and overexpressed in elevated in 50% of primary and 90% of metastatic breast tumors, but is low or absent in normal adult breast tissue. In addition, high Jab1 expression is associated with short progression-free survival durations in breast cancer patients. Our preliminary data showed that inactivation of Jab1/Csn5 in trastuzumab-resistant breast cancer cells sensitized the cells to trastuzumab in a dose- and time-dependent manners. However, the ways in which Jab1 expression is up-regulated in Her2+ breast cancer cells and how this up-regulation drives trastuzumab resistance remain largely unclear. In this study, we will seek to analyze the cross-talk between Src/Stat3 or Akt signaling and Jab1 activation in breast carcinoma. We have found that Stat3 is a novel positive regulator of Jab1 expression in breast cancer cells and that Jab1 overexpression driven by the Src/Stat3 pathway compensates for trastuzumab's inhibition of Her2 signaling in Her2+ breast cancer cells. This suggests that activated Stat3, by up-regulating Jab1 expression, inhibits trastuzumab-induced cell-cycle arrest. In general, our proposed study elucidates Jab1 as a novel contributor to trastuzumab resistance and determine its potential as a prognostic and predictive marker as well as an important therapeutic target. The successful completion of our study would benefit breast cancer patients in three ways. 1) Because Jab1 is rarely expressed in mammary epithelial cells, targeting Jab1 would be less toxic to normal epithelial cells, compared to other agents. 2) Combining Jab1 with trastuzumab would interfere with both Src/Stat3 and Akt pathway. This would significantly benefit patients with Her2+ breast cancer refractory to this therapy due to PTEN loss or activating mutant Stat3. 3) Because Jab1 expression is higher in trastuzumab-resistant cells than in trastuzumab-sensitive cells, Jab1 could be used as a marker of tumor response to trastuzumab. 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 1912. doi:1538-7445.AM2012-1912

Plasma microRNA 210 levels correlate with sensitivity to trastuzumab and tumor presence in breast cancer patients

Trastuzumab is part of the standard treatment for patients with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer, but not all patients respond to trastuzumab. Altered microRNA (miR) expression levels in cancer cells have been correlated with prognosis and response to chemotherapy. The authors of this report hypothesized that altered miR expression levels in plasma are associated with sensitivity to trastuzumab in patients with HER-2 positive breast cancer. Quantitative reverse transcriptase-polymerase chain reaction was used to analyze plasma samples, including samples from patients with breast cancer who were enrolled in a clinical trial of neoadjuvant trastuzumab-based chemotherapy. Expression levels of miR-210, miR-21, miR-29a, and miR-126 were analyzed according to the type of response (pathologic complete response [n = 18] vs residual disease [n = 11]). MicroRNA expression levels also were compared in trastuzumab-sensitive and trastuzumab-resistant breast cancer cells derived from BT474 cells and in an independent set of preoperative plasma samples (n = 39) and postoperative plasma samples (n = 30) from 43 breast cancer patients who did not receive any treatment. At baseline before patients received neoadjuvant chemotherapy combined with trastuzumab, circulating miR-210 levels were significantly higher in those who had residual disease than in those who achieved a pathologic complete response (P = .0359). The mean expression ratio for miR-210 was significantly higher in trastuzumab-resistant BT474 cells, and miR-210 expression was significantly higher before surgery than after surgery (P = .0297) and in patients whose cancer metastasized to the lymph nodes (P = .0030). Circulating miR-210 levels were associated with trastuzumab sensitivity, tumor presence, and lymph node metastases. These results suggest that plasma miR-210 may be used to predict and perhaps monitor response to therapies that contain trastuzumab.

Potent anti-proliferative effects of metformin on trastuzumab-resistant breast cancer cells via inhibition of erbB2/IGF-1 receptor interactions

We have shown that erbB2 altered breast cancer cells are less sensitive to the anti-proliferative effects of metformin than triple negative cells, and have described the differences of molecular mechanisms of metformin action by tumor subtypes. We hypothesized that metformin may be more effective against trastuzumab-resistant erbB2-overexpressing breast cancer cells because it targets the critical signaling pathways that are altered with resistance. BT474, SKBR3 and derived trastuzumab-resistant sublines BT474-HR20 (HR20) and SKBR3-pool2 (pool2) were used to test this hypothesis. Metformin treatment resulted in significantly more inhibition of proliferation and clonogenicity in resistant sublines. It decreased erbB2/insulin-like growth factor-1 receptor (IGF-1R) complexes (present only in the resistant sublines) without altering erbB2 expression, and reduced the expression and activity of erbB3 and IGF-1R in the trastuzumab-resistant but not parental cells. Trastuzumab-resistant sublines were resistant to rapamycin induced changes in mTOR activity and cell growth. In contrast, both BT474 and HR20 cells were highly sensitive to inhibitors of Src (Dasatinib) and PI-3K (LY294002). The pool2 cells showed higher sensitivity than SKBR3 cells to LY294002, but not Dasatinib. On the basis of these data, metformin appears to be significantly more effective against trastuzumab-resistant as compared to sensitive breast cancer cells. Metformin disrupts erbB2/IGF-1R complexes, erbB3 and IGF-1R expression and activity, as well as Src kinase and/or PI-3K/Akt signaling. This action appears to be independent of mTOR signaling. Our findings provide a rationale to study the effects of metformin on patients with erbB2 positive tumors treated with trastuzumab, with or without resistance.

Highlights of This Issue

As new targeted therapies enter clinical trials in the metastatic setting, using primary tumor signatures for patient selection may have profound outcome implications if biomarkers are discordant between the primary and metastatic site. A discordance between the level of potential biomarkers in primary tumor and metastasis could occur due to alterations in gene expression, different microenvironmental stimuli, stochastic events during metastasis, clonal selection during metastasis, or clonal evolution either at the primary site or in the metastasis. PIK3CA mutation and loss of PTEN expression are being pursued as potential predictors of response to novel PI3K pathway inhibitors. A high level of discordance in PTEN level, PIK3CA mutations, and receptor status between primary tumors and metastases may influence patient selection and response to PI3K-targeted therapies.The activation of insulin-like growth factor-I receptor (IGF-IR) contributes to trastuzumab resistance. To understand the roles of insulin-like growth factor binding proteins (IGFBP) in trastuzumab resistance, Dokmanovic and colleagues characterize IGFBP-2 and IGFBP-3 in trastuzumab-sensitive and trastuzumab-resistant breast cancer cells. The authors show that the high levels of secreted IGFBP-3 are associated with trastuzumabsensitive breast cancer cells, whereas the secretion of IGFBP-2 is upregulated in trastuzumab-resistant breast cancer cells. Changes in secretion profiles of IGFBP-2 and IGFBP-3 warrant further clinical investigations as potential biomarkers for trastuzumab resistance.Targeted nanoparticles represent an approach to minimize dose-limiting toxicities associated with chemotherapeutics. A study by Murphy and colleagues describes the design and characterization of a versatile nanoparticle platform, described as nanogels, that combines a lipid bilayer with a cross-linked gel core. The nanogels load a wide array of chemotypes and imaging agents for use as both drug delivery systems and diagnostic tools. The nanogels improve efficacy beyond Abraxane, a clinically approved nanoparticle formulation, in murine orthotopic pancreas and breast cancer models, thereby demonstrating the benefits of targeted nanotherapies in treating malignant disease.Osteosarcoma is an aggressive, often metastatic bone cancer. Toward the goal of developing new treatments for osteosarcoma, Fu and colleagues show apoptosis of osteosarcoma cells by SCH 727965 (SCH), a new cyclindependent kinase inhibitor. Osteosarcoma cell lines resistant to doxorubicin and dasatinib, osteosarcoma cell lines prepared by the authors from patients who had received adjuvant chemotherapy, and osteosarcoma explants derived from a human xenograft in mice were responsive to SCH 727965. Apoptosis occurred at SCH concentrations of 10–20 nM and was p53-independent. SCH did not induce the apoptosis of normal osteoblasts or fibroblasts. Thus, SCH is a promising anticancer agent for osteosarcoma.