HER2-negative Breast Cancer Cells Research Articles

Cell surface interaction of annexin A2 and galectin-3 modulates epidermal growth factor receptor signaling in Her-2 negative breast cancer cells

Overexpression and activation of tyrosine kinase receptors like EGFR and Src regulate the progression and metastasis of Her-2 negative breast cancer. Recently we have reported the role of cell membrane interaction of phospholipid-binding protein annexin A2 (AnxA2) and EGFR in regulating cellular signaling in the activation of angiogenesis, matrix degradation, invasion, and cancer metastasis. Beta-galactoside-specific animal lectin galectin-3 is an apoptosis inhibitor, and cell surface-associated extracellular galectin-3 also has a role in cell migration, cancer progression, and metastasis. Similar expression pattern and membrane co-localization of these two proteins made us to hypothesize in the current study that galectin-3 and AnxA2 interaction is critical for Her-2 negative breast cancer progression. By various experimental analyses, we confirm that glycosylated AnxA2 at the membrane surface interacts with galectin-3. N-linked glycosylation inhibitor tunicamycin treatment convincingly blocked AnxA2 membrane translocation and its association with galectin-3. To analyze whether this interaction has any functional relevance, we tried to dissociate this interaction with purified plant lectin from chickpea (Cicer arietinum agglutinin). This highly specific 30kDa plant lectin could dissociate AnxA2 from endogenous lectin galectin-3 interaction at the cell surface. This dissociation could down-regulate Bcl-2 family proteins, cell proliferation, and migration simultaneously triggering cell apoptosis. Targeting this interaction of membrane surface glycoprotein and its animal lectin in Her-2 negative breast cancer may be of therapeutic value.

Biological indicators of response and resistance to PARP inhibition in BRCA wild-type breast cancer.

125 Background: Although PARP inhibitors are currently being tested in clinical trials in breast cancer patients, it is not well understood which subgroup of patients will best respond to such therapy or how to combine anti-PARP therapy to improve therapeutic response. Methods: We assessed the response of a panel of 8 HER2-negative breast cancer cell lines against three PARP inhibitors: veliparib, olaparib, and BMN 673. Efficacy of carboplatin as a single agent and in combination with PARP inhibitors was determined. Therapeutic response and cellular phenotype were elucidated using live-cell imaging, high-content imaging and immunofluorescence. We computed IC50 as the measure of sensitivity to each PARP inhibitor across the panel of cell lines. Cellular phenotype was ascertained by quantifying endpoints for apoptosis and DNA damage. Results: BMN 673 demonstrated the highest potency, with IC50 values in the nanomolar range, while the IC50 values of olaparib and veliparib were in the micromolar range. Drug sensitivity was independent of BRCA mutational status. Biomarkers of apoptosis and DNA damage varied with the PARP inhibitor and across different cell lines. The resistant cell lines could be classified into two groups that varied in DNA damage response and cell death due to DNA damage. A combined DNA damage – cell death (DDD) Response Score was developed and correlated strongly with IC50 values in BRCA wild-type breast cancer cell lines. The DDD Response Score identified cell lines that were highly sensitive to single-agent PARP inhibitors or carboplatin. A Response Score that identified intermediate sensitivity may predict which cell lines may benefit from the combination of anti-PARP therapy and carboplatin. Conclusions: Overall, a spectrum of response to three different PARP inhibitors was identified in a panel of eight breast cancer lines. Cellular phenotype can help classify resistant cancer cell lines. We have developed an experimental approach that may help to inform which breast cancer subtypes may benefit from PARP inhibitors and carboplatin, both as single agents, and in combination.

Abstract 737: Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance

Abstract Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a subset of pre-treatment specimens from TBCRC006. Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (>100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed. Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ∼30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ∼30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC50: Afa 0.02μM vs. L 3 μM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation. Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura Heiser, Nicholas Wang, Kyle Covington, Edward Chen, Alexander Renwick, Tamika Mitchell, Marty Shea, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne Fuqua, Gary Chamness, Chad Shaw, Marilyn Li, David Wheeler, Susan Hilsenbeck, Mothaffar Fahed Rimawi, Joe Gray, C.Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 737. doi:10.1158/1538-7445.AM2015-737

Abstract P5-05-03: Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib

Abstract Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-03.

Trogocytosis-mediated expression of HER2 on immune cells may be associated with a pathological complete response to trastuzumab-based primary systemic therapy in HER2-overexpressing breast cancer patients

BackgroundTrogocytosis is defined as the transfer of cell-surface membrane proteins and membrane patches from one cell to another through contact. It is reported that human epidermal growth factor receptor 2 (HER2) could be transferred from cancer cells to monocytes via trogocytosis; however, the clinical significance of this is unknown. The aim of this study is to demonstrate the presence and evaluate the clinical significance of HER2+ tumor-infiltrated immune cells (arising through HER2 trogocytosis) in HER2-overexpressing (HER2+) breast cancer patients receiving trastuzumab-based primary systemic therapy (PST).MethodsTo assess the trogocytosis of HER2 from cancer cells to immune cells, and to evaluate the up- and down-regulation of HER2 on immune and cancer cells, peripheral blood mononuclear cells from healthy volunteers and breast cancer patients were co-cultured with HER2+ and HER2-negative breast cancer cell lines with and without trastuzumab, respectively. The correlation between HER2 expression on tumor-infiltrated immune cells and a pathological complete response (pCR) in HER2+ breast cancer patients treated with trastuzumab-based PST was analyzed.ResultsHER2 was transferred from HER2+ breast cancer cells to monocytes and natural killer cells by trogocytosis. Trastuzumab-mediated trogocytosed-HER2+ effector cells exhibited greater CD107a expression than non-HER2-trogocytosed effector cells. In breast cancer patients, HER2 expression on tumor-infiltrated immune cells in treatment naïve HER2+ tumors was associated with a pCR to trastuzumab-based PST.ConclusionsHER2-trogocytosis is visible evidence of tumor microenvironment interaction between cancer cells and immune cells. Given that effective contact between these cells is critical for immune destruction of target cancer cells, this interaction is of great significance. It is possible that HER2 trogocytosis could be used as a predictive biomarker for trastuzumab-based PST efficacy in HER2+ breast cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1041-3) contains supplementary material, which is available to authorized users.

Viral transduction of the HER2-extracellular domain expands trastuzumab-based photoimmunotherapy for HER2-negative breast cancer cells.

The prognosis of HER2-positive breast cancer has been improved by trastuzumab therapy, which features high specificity and limited side effects. However, trastuzumab-based therapy has shortcomings. Firstly, HER2-targeted therapy is only applicable to HER2-expressing tumors, which comprise only 20-25% of primary breast cancers. Secondly, many patients who initially respond to trastuzumab ultimately develop disease progression. To overcome these problems, we employed virus-mediated HER2 transduction and photoimmunotherapy (PIT) which involves trastuzumab conjugated with a photosensitizer, trastuzumab-IR700, and irradiation of near-infrared light. We hypothesized that the gene transduction technique together with PIT would expand the range of tumor entities suitable for trastuzumab-based therapy and improve its antitumor activity. The HER2-extracellular domain (ECD) was transduced by the adenoviral vector, Ad-HER2-ECD, and PIT with trastuzumab-IR700 was applied in the HER2-negative cancer cells. Ad-HER2-ECD can efficiently transduce HER2-ECD into HER2-negative human cancer cells. PIT with trastuzumab-IR700 induced direct cell membrane destruction of Ad-HER2-ECD-transduced HER2-negative cancer cells. Novel combination of viral transduction of a target antigen and an antibody-based PIT would expand and potentiate molecular-targeted therapy even for target-negative or attenuated cancer cells.

Sticky Patches on Lipid Nanoparticles Generate Binding Geometries that Enable Effective Targeting of Otherwise Untargetable Cancers

The majority of breast cancer patients (70%) have tumors designated as ‘HER2-negative’ (<1+ HER2-expression evaluated by immunohistochemistry or < 200,000 HER2-copies per cell). For these patients there are no targeted therapeutic options utilizing the HER2 receptor. The ability of conventionally targeted nanoparticles for specific targeting stops to hold on cancer cells expressing less than 200,000 copies of HER2 per cell or less than two receptors per nanoparticle's projected area (for particles of 100 nm in-diameter). This geometry corresponds to the limit of multivalent interactions (avidity) loosely defined as multiple contacts between neighboring same-cell receptors with ligands from a single nanoparticle. An alternative therapeutic approach is needed, therefore, to enable selective targeting and effective killing of cancer cells with low or too low HER2 expression.Towards this goal we designed targeted lipid nanoparticles (vesicles) that contain HER2-targeting short peptides densely conjugated (for high local multivalency) within sticky patches. Sticky patches are phase-separated raft-like lipid-domains of high local multivalency which is induced by preferential partitioning of peptide-functionalized lipids. To enable selectivity in binding, sticky patches are exclusively triggered to form in mildly acidic environments matching the tumor interstitium. Lipid phase-separation with lowering pH is a result of the interplay of decreasing (pH-tunable) electrostatic repulsion and attractive hydrogen bonding among the domain-forming lipids.We show that lipid nanoparticles with sticky patches selectively associate with and kill HER2-negative and triple negative breast cancer cells (MCF-7 and MDA-MB-231, respectively, with 60,000 and 90,000 HER2-copies per cell) while do not affect cardiomyocytes and breast normal cells. Systematic studies of association, dissociation and internalization rates of nanoparticles by cells will be presented, and a mechanistic mathematical model will be discussed with the aim to explain the observed high avidity.

Abstract 2615: Novel combination therapy of adenoviral gene transfer of HER2-extracellular domain and trastuzumab-based photoimmunotherapy for HER2 negative cancer cells

Abstract Introduction: Humanized anti-HER2 monoclonal antibody, trastuzumab, has provided survival benefit to patients with HER2 positive breast cancer and advanced gastric cancer. However, the treatment with trastuzumab was limited due to low or heterogeneous HER2 positivity, insufficient effects as single agent and acquisition of resistance. To overcome these problems, we employed the adenoviral vector expressing HER2-extracellular domain (HER2-ECD), Ad/ HER2-ECD, which is designed to tag cancer cells with HER2-ECD and to sensitize them to trastuzumab, and a novel photoimmunotherapy (PIT). PIT consists of trastuzumab-conjugated with a photosensitizer, IR700 (Tmab-IR700) and irradiation with near-infrared light, induces a strong cytotoxicity on HER2 positive cancer cells. We assessed the effect of Ad/HER2-ECD and Tmab-IR700 mediated PIT in HER2 negative cancer cells. Methods: HER2 negative breast cancer cells (MCF7, MDA-MB-231) and gastric cancer cells (MKN1 and MKN45) were infected with Ad/ HER2-ECD. They were treated with Tmab-IR700 mediated PIT. We evaluated the HER2-ECD expression induced by Ad/ HER2-ECD and the cytotoxic effects of Tmab-IR700 mediated PIT. Results: Ad/HER2-ECD efficiently expressed HER2 protein on the surface of the cancer cells. HER2-ECD expressing cells were selectively and rapidly killed by Tmab-IR700 mediated PIT, which was morphologically captured as bleb formation of cellular membrane. Conclusions: We demonstrated that Ad/ HER-ECD combined with Tmab-IR700 mediated PIT could induce target-selective, immediate cell death in HER2 negative cancer cells. The combination therapy of adenoviral gene transfer of HER2-ECD and the molecular-target PIT is effective for cancer cells regardless of HER2 expression. Citation Format: Michihiro Ishida, Shunsuke Kagawa, Kyoko Shimoyama, Kiyoto Takehara, Kazuhiro Noma, Shunsuke Tanabe, Hiroshi Tazawa, Junji Matsuoka, Hisataka Kobayashi, Toshiyoshi Fujiwara. Novel combination therapy of adenoviral gene transfer of HER2-extracellular domain and trastuzumab-based photoimmunotherapy for HER2 negative cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2615. doi:10.1158/1538-7445.AM2014-2615

Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (T-DM1)

The antibody trastuzumab (Herceptin) has substantially improved overall survival for patients with aggressive HER2-positive breast cancer. However, about 70% of all treated patients will experience relapse or disease progression. This may be related to an insufficient targeting of the CD44highCD24low breast cancer stem cell subset, which is not only highly resistant to chemotherapy and radiotherapy but also a poor target for trastuzumab due to low HER2 surface expression. Hence, we explored whether the new antibody-drug conjugate T-DM1, which consists of the potent chemotherapeutic DM1 coupled to trastuzumab, could improve the targeting of these tumor-initiating or metastasis-initiating cells. To this aim, primary HER2-overexpressing tumor cells as well as HER2-positive and HER2-negative breast cancer cell lines were treated with T-DM1, and effects on survival, colony formation, gene and protein expression as well as antibody internalization were assessed. This revealed that CD44highCD24lowHER2low stem cell-like breast cancer cells show high endocytic activity and are thus particularly sensitive towards the antibody-drug conjugate T-DM1. Consequently, preexisting CD44highCD24low cancer stem cells were depleted by concentrations of T-DM1 that did not affect the bulk of the tumor cells. Likewise, colony formation was efficiently suppressed. Moreover, when tumor cells were cocultured with natural killer cells, antibody-dependent cell-mediated cytotoxicity was enhanced, and EMT-mediated induction of stem cell-like properties was prevented in differentiated tumor cells. Thus our study reveals an unanticipated targeting of stem cell-like breast cancer cells by T-DM1 that may contribute to the clinical efficacy of this recently approved antibody-drug conjugate.

Trichostatin A Suppresses EGFR Expression through Induction of MicroRNA-7 in an HDAC-Independent Manner in Lapatinib-Treated Cells

Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, has been shown to improve the survival rate of patients with advanced HER2-positive breast cancers. However, the off-target activity of lapatinib in inducing EGFR expression without tyrosine kinase activity was demonstrated to render HER2-negative breast cancer cells more metastatic, suggesting a limitation to the therapeutic effectiveness of this dual inhibitor in HER2-heterogeneous tumors. Therefore, targeting EGFR expression may be a feasible approach to improve the anticancer efficiency of lapatinib-based therapy. Inhibition of HDAC has been previously reported to epigenetically suppress EGFR protein expression. In this study, however, our data indicated that treatment with HDAC inhibitors trichostatin A (TSA), but not suberoylanilide hydroxamic acid (SAHA) or HDAC siRNA, can attenuate both protein and mRNA expressions of EGFR in lapatinib-treated triple-negative breast cancer cells, suggesting that TSA may suppress EGFR expression independently of HDAC inhibition. Nevertheless, TSA reduced EGFR 3′UTR activity and induced the gene expression of microRNA-7, a known EGFR-targeting microRNA. Furthermore, treatment with microRNA-7 inhibitor attenuated TSA-mediated EGFR suppression. These results suggest that TSA induced microRNA-7 expression to downregulate EGFR expression in an HDAC-independent manner.

Abstract P5-08-06: PI3K blockade enhances the antitumor activity of eribulin in PIK3CA-mutant eribulin-resistant tumor xenografts

Abstract Eribulin is a recently approved microtubule-targeting agent (MTA) for the management of heavily pretreated metastatic breast cancer (BC) patients. Constitutive PI3K/Akt/mTOR survival pathway activation, either via mutational activation of the PI3K catalytic subunit (PIK3CA) or via inactivation of the tumor suppressor PTEN, may confer resistance to MTAs. Hence, we hypothesized that PI3K-pathway activation limits the antitumor activity of eribulin in HER2-negative BC and that PI3K inhibition enhances the efficacy of this chemotherapeutic agent. The predictive value of PIK3CA mutation or PTEN loss towards eribulin response was interrogated in vitro, using a panel of fourteen HER2-negative BC cell lines, and in vivo using six tumor models from cell-line or patient-derived tumors. While PIK3CA mutation did not seem to be predictive in vitro, the PIK3CA-mutated xenograft models underwent tumor progression upon single-agent eribulin therapy (Table 1). In the absence of concomitant PIK3CA mutation, PTEN loss was neither predictive in vitro nor in vivo. Moreover, eribulin induced PI3K-pathway activation in tumor xenografts, a potential escape mechanism to MTA-therapy. To validate the negative predictive value of PIK3CA mutation, BC tumors from patients treated with eribulin in the neoadjuvant and in the metastatic setting are being queried, as the pharmacodynamic activation of the PI3K-pathway upon eribulin treatment. Class I pan-PI3K (BKM120) or PI3K-a-specific (BYL719) inhibitors were used in vitro to block the PI3K-pathway concomitantly with eribulin treatment, resulting in enhanced antiproliferative and proapoptotic activity. Strikingly, in PIK3CA mutant xenograft models, while eribulin alone exhibited limited antitumor activity compared to PIK3CA-wild type models, co-administration of a PI3K inhibitor induced marked tumor regression (BKM120 data in Table 1, BYL719 data will be reported). Moreover, addition of the PI3K inhibitor at progression with eribulin single-agent also resulted in tumor regression. Of note, PIK3CA-wild type models also exhibited increased antitumor activity with the combined therapy compared to single-agent treatments. The precise mechanism by which the combination of eribulin and a PI3K-targeting agent results in tumor regression is currently under investigation, embracing both the induction of mitotic catastrophe in tumor cells and the regularization of the tumor vasculature. These results support the clinical development of therapeutic regimens combining PI3K-inhibitors to the approved MTA eribulin and might be predictive of clinical benefit both in the PIK3CA-mutant and -wild type breast cancer population. Table 1. Percentage change in tumor volume.StatusPIK3CA mutantPIK3CA mutant and PTEN-lowPTEN-lowWTModel/ TreatmentMCF7LPDX44CAL51MDA-MB-468PDX88PDX98Eribulin39 ± 9683 ± 51364 ± 127-47 ± 14-26 ± 34-36 ± 33BKM120501 ± 169116 ± 111119 ± 73-9 ± 23128 ± 3787 ± 112Eribulin + BKM120-70 ± 27-80 ± 1051 ± 46-75 ± 12-94 ± 5-65 ± 13Patient- (PDX) and cell line-derived tumor xenografts were treated with eribulin mesylate (0.1mg/kg, 3IW) and/or BKM120 (27.5 mg/kg, 6IW) for 26-31 days.. IW, in week. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-06.

BAY 80-6946 Is a Highly Selective Intravenous PI3K Inhibitor with Potent p110α and p110δ Activities in Tumor Cell Lines and Xenograft Models

Because of the complexity derived from the existence of various phosphoinositide 3-kinase (PI3K) isoforms and their differential roles in cancers, development of PI3K inhibitors with differential pharmacologic and pharmacokinetic profiles would allow best exploration in different indications, combinations, and dosing regimens. Here, we report BAY 80-6946, a highly selective and potent pan-class I PI3K inhibitor with sub-nanomolar IC50s against PI3Kα and PI3Kδ. BAY 80-6946 exhibited preferential inhibition (about 10-fold) of AKT phosphorylation by PI3Kα compared with PI3Kβ in cells. BAY 80-6946 showed superior antitumor activity (>40-fold) in PIK3CA mutant and/or HER2 overexpression as compared with HER2-negative and wild-type PIK3CA breast cancer cell lines. In addition, BAY 80-6946 revealed potent activity to induce apoptosis in a subset of tumor cells with aberrant activation of PI3K as a single agent. In vivo, single intravenous administration of BAY 80-6946 exhibited higher exposure and prolonged inhibition of pAKT levels in tumors versus plasma. BAY 80-6946 is efficacious in tumors with activated PI3K when dosed either continuously or intermittently. Thus, BAY 80-6946 induced 100% complete tumor regression when dosed as a single agent every second day in rats bearing HER2-amplified and PIK3CA-mutated KPL4 breast tumors. In combination with paclitaxel, weekly dosing of BAY 80-6946 is sufficient to reach sustained response in all animals bearing patient-derived non-small cell lung cancer xenografts, despite a short plasma elimination half-life (1 hour) in mice. Thus, BAY 80-6946 is a promising agent with differential pharmacologic and pharmacokinetic properties for the treatment of PI3K-dependent human tumors.

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

Dithiiranylmethyloxy azaxanthone (CHO10), which was discovered by screening compounds in a reporter gene assay, inhibited the ESX–Sur2 interaction in a dose-dependent manner with potency similar to canertinib. The intervention of CHO10 during the ESX–Sur2 interaction caused down-regulation of both HER2 gene amplification and HER2 protein expression, which led to the attenuation of HER2-mediated downstream signal cascades and autocrine cell growth in SK-BR-3 cells, which are HER2 overexpressing breast cancer cells. The cell growth inhibitory activity of CHO10 was more potent in HER2-overexpressing breast cancer cells (AU-565, BT474 and SK-BR-3) than in HER2-negative cells (HEK293) and breast cancer cells (MCF-7) that express a basal level of HER2. Treatment with CHO10 in combination with tamoxifen sensitized BT474 cells, tamoxifen-resistant ER-positive breast cancer cell line, toward chemotherapeutic. The anti-tumor activity of CHO10 was validated by the significant reduction in tumor size of NCI-H460 or DLD-1 subcutaneously implanted xenograft tumors through treatment with 1mg/kg five times every other 2days.

A Targeted RNAi Screen of the Breast Cancer Genome Identifies KIF14 and TLN1 as Genes That Modulate Docetaxel Chemosensitivity in Triple-Negative Breast Cancer

To identify biomarkers within the breast cancer genome that may predict chemosensitivity in breast cancer. We conducted an RNA interference (RNAi) screen within the breast cancer genome for genes whose loss-of-function enhanced docetaxel chemosensitivity in an estrogen receptor-negative, progesterone receptor-negative, and Her2-negative (ER-, PR-, and Her2-, respectively) breast cancer cell line, MDA-MB-231. Top candidates were tested for their ability to modulate chemosensitivity in 8 breast cancer cell lines and to show in vivo chemosensitivity in a mouse xenograft model. From ranking chemosensitivity of 328 short hairpin RNA (shRNA) MDA-MB-231 cell lines (targeting 133 genes with known somatic mutations in breast cancer), we focused on the top two genes, kinesin family member 14 (KIF14) and talin 1 (TLN1). KIF14 and TLN1 loss-of-function significantly enhanced chemosensitivity in four triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, HCC38, HCC1937, and Hs478T) but not in three hormone receptor-positive cell lines (MCF7, T47D, and HCC1428) or normal human mammary epithelial cells (HMEC). Decreased expression of KIF14, but not TLN1, also enhanced docetaxel sensitivity in a Her2-amplified breast cancer cell line, SUM190PT. Higher KIF14 and TLN1 expressions are found in TNBCs compared with the other clinical subtypes. Mammary fat pad xenografts of KIF14- and TLN1-deficient MDA-MB-231 cells revealed reduced tumor mass compared with control MDA-MB-231 cells after chemotherapy. KIF14 expression is also prognostic of relapse-free and overall survival in representative breast cancer expression arrays. KIF14 and TLN1 are modulators of response to docetaxel and potential therapeutic targets in TNBC.

Active targeting of HER2-positive breast cancer cells by Herceptin-functionalized organically modified silica nanoparticles.

Normal micelle microemulsion method was utilized for fabrication of organically modified silica (ORMOSIL) nanoparticles. The void and dye-doped nanoparticles were synthesized in nonpolar core of two different surfactants including Aerosol OT and Tween 80. The nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, and zeta potential analysis. Our results revealed that the type of surfactant molecules has a dramatic impact on the size and size distribution range, surface charge, and surface functionalization of the nanoparticles. The particles fabricated using Tween 80 had very smaller size with narrow size distribution and very lower amount of zeta potential. For specific delivery of functionalized nanoparticles to breast cancer cell line SKBR3, overexpressing human epidermal growth factor receptor 2 (HER2), both dye-doped nanoparticles fabricated with Aerosol OT or Tween 80, was conjugated to Herceptin. In vitro studies using fluorescent microscopy demonstrated that the surfactant used for preparation of the nanoparticles can affect the uptake of the particles by cells. The dye-doped functionalized ORMOSIL nanoparticles prepared with Aerosol OT showed better efficiency in the process of active targeting of HER2 receptor. Herceptin-functionalized ORMOSIL nanoparticles can be used for differentiation of HER2-positive from HER2-negative breast cancer cells or specific delivery of therapeutic and diagnostic agents and also other nanoparticles such as magnetic nanoparticles and quantum dots to breast cancer cells.FigureOrmosil bioconjugation

The role of radiotherapy-resistant stem cells in breast cancer recurrence.

Although the effectiveness of anti cancer therapies has improved dramatically, breast cancer (BC) remains the second leading cause for cancer-related death among women in the western world [1]. The observed improvement in the survival rate and remission for primary tumors are essentially related to the advances in early diagnosis with more sensitive imaging technology [2,3]. However, the death rate remains unacceptably high for BC patients due to hard-to-treat metastatic and recurrent tumors, necessitating new, effective approaches and anticancer agents. Accumulating evidence suggests that cancer stem cells (CSCs), which are present in many cancers including BC, mediate tumor metastasis and contribute to relapse due to their resistance to current conventional therapies [4,5]. These unique cells possess stem cell-like characteristics, such as the capacity of self-renewal, which makes the tumor capable of regenerating its entire bulk. CSCs are resistant to proapoptotic factors, rendering them a formidable adversary to anticancer agents. In part, this is related to their quiescence capacity, which holds them in a standby mode in their niche microenvironment, sheltering them from radiation and other anticancer agents, since these agents are effective only on highly proliferative cells [6,7]. In addition to their powerful DNA repair machinery, breast CSCs (BCSCs) express ALDH, which is suspected to play a part in their death-resistance phenotype by being involved with cell detoxification machinery [8,9]. Thus, BCSCs represent a major challenge in the battle against BC, and an opportunity to develop more effective target to treat metastatic lesions.

HER2-Associated Radioresistance of Breast Cancer Stem Cells Isolated from HER2-Negative Breast Cancer Cells

To understand the role of HER2-associated signaling network in breast cancer stem cells (BCSC) using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2(-/low) breast cancers. HER2-expressing BCSCs (HER2(+)/CD44(+)/CD24(-/low)) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated signaling network was generated by MetaCore program. Compared with HER2-negative BCSCs, HER2(+)/CD44(+)/CD24(-/low) cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2(+)/CD44(+)/CD24(-/low) cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/MS-MS of HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs reported a unique HER2-associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function, and DNA repair. A specific feature of HER2-STAT3 network was identified. This study provides the evidence that HER2-mediated prosurvival signaling network is responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant HER2(-/low) breast cancer.

HER2 Overexpression Renders Human Breast Cancers Sensitive to PARP Inhibition Independently of Any Defect in Homologous Recombination DNA Repair

HER2 overexpression in breast cancer confers increased tumor aggressiveness. Although anti-HER2 therapies have improved patient outcome, resistance ultimately occurs. PARP inhibitors target homologous recombination (HR)-deficient tumors, such as the BRCA-associated breast and ovarian cancers. In this study, we show that HER2+ breast cancers are susceptible to PARP inhibition independent of an HR deficiency. HER2 overexpression in HER2 negative breast cancer cells was sufficient to render cells susceptible to the PARP inhibitors ABT-888 and AZD-2281 both in vitro and in vivo, which was abrogated by HER2 reduction. In addition, ABT-888 significantly inhibited NF-κB (p65/RelA) transcriptional activity in HER2+ but not HER2 negative breast cancer cells. This corresponded with a reduction in phosphorylated p65 and total IKKα levels, with a concomitant increase in IκBα. Overexpression of p65 abrogated cellular sensitivity to ABT-888, whereas IκBα overexpression reduced cell viability to a similar extent as ABT-888. Therefore, susceptibility of HER2+ breast cancer cells to PARP inhibition may be because of inhibition of NF-κB signaling driven by HER2. Our findings indicate that PARP inhibitors may be a novel therapeutic strategy for sporadic HER2+ breast cancer patients.

446PD - Phase I Study of Afatinib (BIBW 2992), An Erbb Family Blocker Plus Nintedanib (BIBF 1120), A Triple Angiokinase Inhibitor, in Patients (PTS) With Advanced Solid Tumours

ABSTRACT Background Inhibiting multiple signalling pathways with the combination of afatinib, an oral irreversible ErbB Family Blocker, and nintedanib, an oral triple angiokinase inhibitor of VEGFR, PDGFR and FGFR, may lead to better efficacy. Methods This Phase I study used a modified 3 + 3 design to determine the maximum tolerated dose (MTD) of afatinib, with dose escalating from 10 to 40 mg qd given in 2 schedules: continuous (C) or intermittent (I) (every other week), in combination with fixed-dose nintedanib (200 mg bid reduced to 150 mg bid after protocol amendment) in a 28-day cycle. Secondary endpoints were safety, efficacy, pharmacokinetics (PK), and circulating tumour cells (CTC) analysis. Treatment continued until disease progression or intolerability. Results 45 pts with heavily pretreated advanced solid tumours were included: 26 men; median age 56 years (range 37–73); main cancer types: lung (NSCLC), colorectal, breast, melanoma and ovary. Main adverse events were diarrhoea, asthenia, nausea, vomiting and transaminase elevation. Two MTDs were established: afatinib 40 mg qd (I) plus nintedanib 150 mg bid and afatinib 30 mg qd (C) plus nintedanib 150 mg bid (Table). Efficacy data showed evidence of antitumour activity with partial responses (RECIST) observed in 2 pts (HER2-negative breast cancer, and head & neck squamous cell carcinoma) and stable disease in 27 pts (lasting >3 months in 8 pts). Preliminary PK data showed no drug–drug interaction. CTC analysis will be presented. Afatinib (C or I) (mg qd) Nintedanib (mg bid) Evaluable pts/dose-limiting toxicity (DLT) DLT/Grade (G) 10 C 200 3/0 20 C 200 3/0 30 C 200 7/2 diarrhoea G3, transaminase elevation G3 40 C 200 3/3 diarrhoea G3, transaminase elevation G3; creatinine increase G2 30 I 200 6/2 diarrhoea G3, transaminase elevation G3, creatinine increase G2 40 I 200 6/2 dehydration G3, transaminase elevation G4 40 C 150 3/2 diarrhoea G3, renal failure G3 40 I 150 6/0 MTD 30 C 150 6/0 MTD Conclusion At MTD, the combination of afatinib with nintedanib showed a manageable safety profile and evidence of activity in different heavily pretreated tumour types. Disclosure J. Soria: Honoraria from Boehringer Ingelheim and Roche. N. Morsli: Employee of Boehringer Ingelheim. M. Ould Kaci: Employee of Boehringer Ingelheim. H. Staines: Employee of Boehringer Ingelheim. K. Marzin: Employee of Boehringer Ingelheim. All other authors have declared no conflicts of interest.

Novel HER2 Aptamer Selectively Delivers Cytotoxic Drug to HER2-positive Breast Cancer Cells in Vitro

BackgroundAptamer-based tumor targeted drug delivery system is a promising approach that may increase the efficacy of chemotherapy and reduce the related toxicity. HER2 protein is an attractive target for tumor-specific drug delivery because of its overexpression in multiple malignancies, including breast, gastric, ovarian, and lung cancers.MethodsIn this paper, we developed a new HER2 aptamer (HB5) by using systematic evolution of ligands by exponential enrichment technology (SELEX) and exploited its role as a targeting ligand for delivering doxorubicin (Dox) to breast cancer cells in vitro.ResultsThe selected aptamer was an 86-nucleotide DNA molecule that bound to an epitope peptide of HER2 with a Kd of 18.9 nM. The aptamer also bound to the extracellular domain (ECD) of HER2 protein with a Kdof 316 nM, and had minimal cross reactivity to albumin or trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative breast cancer cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating Dox into the DNA structure of HB5. The Apt-Dox complex could selectively deliver Dox to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells in vitro. Moreover, Apt-Dox retained the cytotoxicity of Dox against HER2-positive breast cancer cells, but reduced the cytotoxicity to HER2-negative cells.ConclusionsThe results suggest that the selected HER2 aptamer may have application potentials in targeted therapy against HER2-positive breast cancer cells.