Trastuzumab-resistant Cells Research Articles

Trastuzumab stimulation of ribosomal protein S6 kinase 1 (S6K1) predicts de novo trastuzumab resistance

Breast cancer is a complex disease with at least five different molecular subtypes identified. The breast tumor molecular subtypes guide stratification of patients for specific targeted therapy regimens and each subtype is associated with significantly different patient outcomes. For example, patients with the HER2 positive molecular subtype benefit from the HER2 targeted therapy trastuzumab. Unfortunately, women with the HER2 positive molecular subtype have the worst overall prognosis and nearly 70% of women with HER2 positive breast cancer exhibit de novo or acquired resistance to trastuzumab. Identification of tumor markers predicting trastuzumab response can be used to further stratify patients for life-saving personalized therapeutic options. The aim of this study was to identify clinically useful tumor markers predicting de novo tumor cell resistance to trastuzumab treatment. To identify oncogenic signaling pathways activated in response to trastuzumab treatment, we performed a Human Phospho-Kinase Proteome Profiler Array analysis comparing trastuzumab sensitive MCF-7/HER2.2 and trastuzumab resistant MCF-7/HER2Δ16H cells following acute treatment with 20 μg/ml of trastuzumab for 2 h. We found that of the 43 phosphorylation activated human kinases represented on the array, S6K1 was the only kinase altered greater than 1.5-fold in response to trastuzumab treatment of the trastuzumab resistant MCF-7/HER2Δ16H cells. Trastuzumab activation of S6K1 was confirmed in the two trastuzumab resistant SUM190 and SUM225 cell lines. Significantly, trastuzumab failed to stimulate S6K1 activation in the trastuzumab sensitive MCF-7/HER2.2, BT474, and SKBR3 cell lines suggesting that trastuzumab activation of S6K1 is a tumor cell marker for trastuzumab resistance. Consistent with a role for mTORC1/S6K1 signaling promoting trastuzumab resistance, all cell lines were sensitive to S6K1 inactivation with significant growth inhibition following treatment with the mTORC1 inhibitor rapamycin. In conclusion, characterizing rapid trastuzumab induced molecular alterations resulted in the identification of activated S6K1 as an early breast tumor cell marker for trastuzumab resistance. Our results further suggest that trastuzumab resistant breast tumor cells are addicted to mTORC1/S6K1 oncogenic signaling and targeting mTORC1 with rapamycin reverses trastuzumab resistance.

An FGFR3 Autocrine Loop Sustains Acquired Resistance to Trastuzumab in Gastric Cancer Patients.

The majority of gastric cancer patients who achieve an initial response to trastuzumab-based regimens develop resistance within 1 year of treatment. This study was aimed at identifying the molecular mechanisms responsible for resistance. A HER2+-trastuzumab sensitive NCI-N87 gastric cancer orthotopic nude mouse model was treated with trastuzumab until resistance emerged. Differentially expressed transcripts between trastuzumab-resistant and sensitive gastric cancer cell lines were annotated for functional interrelatedness by Ingenuity Pathway Analysis software. Immunohistochemical analyses were performed in pretreatment versus posttreatment biopsies from gastric cancer patients receiving trastuzumab-based treatments. All statistical tests were two-sided. Four NCI-N87 trastuzumab-resistant (N87-TR) cell lines were established. Microarray analysis showed HER2 downregulation, induction of epithelial-to-mesenchymal transition, and indicated fibroblast growth factor receptor 3 (FGFR3) as one of the top upregulated genes in N87-TR cell lines. In vitro, N87-TR cell lines demonstrated a higher sensitivity than did trastuzumab-sensitive parental cells to the FGFR3 inhibitor dovitinib, which reduced expression of pAKT, ZEB1, and cell migration. Oral dovitinib significantly (P = 0.0006) reduced tumor burden and prolonged mice survival duration in N87-TR mouse models. A higher expression of FGFR3, phosphorylated AKT, and ZEB1 were observed in biopsies from patients progressing under trastuzumab-based therapies if compared with matched pretreatment biopsies. This study identified the FGFR3/AKT axis as an escape pathway responsible for trastuzumab resistance in gastric cancer, thus indicating the inhibition of FGFR3 as a potential strategy to modulate this resistance. Clin Cancer Res; 22(24); 6164-75. ©2016 AACR.

Fucose-containing fraction of Ling-Zhi enhances lipid rafts-dependent ubiquitination of TGFβ receptor degradation and attenuates breast cancer tumorigenesis.

Ganoderma lucidum exerts antitumor activity, but the mechanism of G. lucidum polysaccharides on cancer is unclear. Here, we demonstrated that a fucose-containing fraction of Ling-Zhi (FFLZ) reduced tumor size and suppressed metastasis in vivo. Furthermore, FFLZ inhibited breast cancer cell migration and altered the epithelial-to-mesenchymal transition (EMT) phenotype. Transforming growth factor-β receptor (TGFR) pathways act as key mediators to promote tumor progression and metastasis. We found that FFLZ down-regulated TGFR and downstream signaling pathways, including the phosphorylation of Smad2/3 and the expression of Smad4. In an investigation of the underlying mechanisms, we found that FFLZ enhanced the Smurf2-dependent ubiquitination of TGFR by disrupting the balance of the lipid rafts, promoted the “re-localization” of the TGFR to the caveolae, and facilitated the degradation of TGFR. Together, our data indicated that FFLZ is associated with the inhibition of EMT and the prevention of metastasis by promoting ubiquitination-dependent TGFR degradation and abolishing TGFR signaling pathways. Moreover, the combination of FFLZ and trastuzumab synergistically inhibited the viability of certain trastuzumab-resistant human breast cancer cells. In summary, our current findings indicate that FFLZ is a potential therapeutic or dietary supplemental agent for cancer patients and that it functions via the caveolin-1/Smad7/Smurf2-dependent ubiquitin-mediated degradation of TGFR.

Identification of KLK10 as a therapeutic target to reverse trastuzumab resistance in breast cancer.

Trastuzumab, the first antibody widely used in anti-HER2 targeted therapy, dramatically improved the overall outcome of HER2 positive breast cancer patients. However, trastuzumab resistance emerged as a major problem in its clinical application. In order to explore mechanisms underlying trastuzumab resistance, we performed RNA-Seq to analyze the gene expression variation in trastuzumab resistant breast cancer cell line. The sequencing result was then combined with the relevant data in TCGA database to conduct a co-expression analysis. We found a series of differentially expressed genes with potential contributions to trastuzumab resistance. Among them, KLK10 was verified to be a potential therapeutic target for reversing trastuzumab resistance. In summary, this study provides a new clue to screen molecular targets and predictive biomarkers for trastuzumab resistance.

Abstract B069: The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC

Abstract Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab')2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies.Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be.The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition. Citation Format: Gábor Tóth, Árpád Szöőr, László Simon, Yosef Yarden, János Szöllősi, György Vereb. The best is not enough—when it is alone: Current clinical doses of humanized anti-Her2 antibodies should be applied in combination to delay development of trastuzumab resistance by additively enhancing ADCC [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B069.

Sensitizing effect of juglone is mediated by down regulation of Notch1 signaling pathway in trastuzumab-resistant SKBR3 cells.

Trastuzumab (Herceptin) monoclonal antibody directed against HER2 receptor has been administered as a treatment for metastatic HER2 positive breast cancer. The problematic issue in treatment of HER2 positive breast cancer cells is commonly the induction of resistance to trastuzumab which might be due to modulation of some vital signaling elements such as Notch1 and Pin1. In this study, we were aimed to investigate whether the cross talk between pin1 and Notch1 has a role in this event. Our results indicated that the expression level of Pin1 in resistant SKBR3 cells increased by about twofold relative to sensitive SKBR3 cells. Besides, Pin1 inhibition via juglone reduced the extent of proliferation, colony formation and migration capacity of resistant SKBR3 cells. In addition, despite a feed forward loop between Notch1 and Pin1 in sensitive SKBR3 cells, inhibition of Notch1 cleavage in resistant SKBR3 cells did not affect pin1 level whereas pin1 inhibition by juglone reduced the level of Hes1, p-Akt and increased the cellular content of Numb. Therefore, we concluded that pin1 inhibition could be considered as a promising sensitizing strategy to weaken trastuzumab resistance.

Combined SRC inhibitor saracatinib and anti-ErbB2 antibody H2-18 produces a synergistic antitumor effect on trastuzumab-resistant breast cancer

Despite of the effectiveness of the anti-ErbB2 humanized antibody trastuzumab, trastuzumab resistance emerges as a major and common clinical problem. Thus, circumventing trastuzumab resistance has become an urgent need. Recently, Src inhibitor saracatinib has drawn great attention for its key role in trastuzumab response. As shown in our previous study, H2-18, an anti-ErbB2 antibody, could potently induce programmed cell death (PCD) in trastuzumab-resistant breast cancer cells. Here we combined H2-18 and a Src inhibitor, saracatinib, and studied the antitumor activity of this drug combination in trastuzumab-resistant breast cancer cell lines. The results showed that H2-18 and saracatinib could synergistically inhibit cell proliferation of BT-474, SKBR-3, HCC-1954 and HCC-1419 breast cancer cell lines in vitro. H2-18 plus saracatinib could also inhibit the HCC-1954 tumor growth more effectively in vivo than each drug alone. H2-18 plus saracatinib showed a significantly more potent PCD-inducing activity compared with either H2-18 or saracatinib alone. We conclude that enhanced PCD may contribute to the superior antitumor efficacy of this combination therapy. The combination of H2-18 and SRC inhibitor has the potential to be translated into clinic.

Mycophenolic acid potentiates HER2-overexpressing SKBR3 breast cancer cell line to induce apoptosis: involvement of AKT/FOXO1 and JAK2/STAT3 pathways.

Trastuzumab has been successfully used as a first-line therapy specific for HER2-overexressing breast cancer patients. However, despite the effectiveness of trastuzumab, the occurrence of inherent and acquired resistance remains as the main challenge of the therapy. Thus, this has motivated efforts toward finding new therapeutic strategies including combining trastuzumab with other drugs to enhance its therapeutic efficacy. In that line, we investigated the capability of mycophenolic acid (MPA), an inhibitor of de novo guanine nucleotide synthesis with potential anti-cancer activity, on improving the response to trastuzumab among SKBR3 cells as well as trastuzumab resistant SKBR3-TR cells. Our data indicated that irrespective to trastuzumab sensitivity of cells, MPA effectively inhibited cell growth through inducing adipocyte-like cell differentiation as well as blocking cell cycle progression at G1 phase along with augmentation of p27kip expression level. Furthermore, combined treatment with trastuzumab and MPA was more potent in cell growth inhibition, cell cycle arrest and apoptosis induction, as evident by flow cytometric analyses and caspase-3 production, in both trastuzumab sensitive and resistant SKBR3 cells. Besides, western blot analysis showed that elevated apoptosis induction in both cell groups was associated with attenuation in phosphorylation of some key elements of HER2 signaling pathway including AKT, ERK, STAT3 and consequently augmentation in FOXO1 expression level in response to combination of trastuzumab and MPA. These data suggest that manipulation of intracellular GTP level by MPA and consequent molecular perturbation in some of the cell survival and pro-apoptotic relevant signaling pathways might provide an alternative clinical strategy for chemosensitization of resistant breast cancer cells to anti- HER2 therapy.

B14 - The combination of GDC-0980, a PI3K/mTOR kinase inhibitor and BAY-86-9766, a MEK inhibitor is able to induce cell growth inhibition of HER2 positive Trastuzumab resistant gastric cancer cell lines

B14 - The combination of GDC-0980, a PI3K/mTOR kinase inhibitor and BAY-86-9766, a MEK inhibitor is able to induce cell growth inhibition of HER2 positive Trastuzumab resistant gastric cancer cell lines

Functionalized immunostimulating complexes with protein A via lipid vinyl sulfones to deliver cancer drugs to trastuzumab-resistant HER2-overexpressing breast cancer cells.

BackgroundAround 20%–30% of breast cancers overexpress the proto-oncogene human epidermal growth receptor 2 (HER2), and they are characterized by being very invasive. Therefore, many current studies are focused on testing new therapies against tumors that overexpress this receptor. In particular, there exists major interest in new strategies to fight breast cancer resistant to trastuzumab (Tmab), a humanized antibody that binds specifically to HER2 interfering with its mitogenic signaling. Our team has previously developed immunostimulating complexes (ISCOMs) as nanocapsules functionalized with lipid vinyl sulfones, which can incorporate protein A and bind to G immunoglobulins that makes them very flexible nanocarriers.Methods and resultsThe aim of this in vitro study was to synthesize and evaluate a drug delivery system based on protein A-functionalized ISCOMs to target HER2-overexpressing cells. We describe the preparation of ISCOMs, the loading with the drugs doxorubicin and paclitaxel, the binding of ISCOMs to alkyl vinyl sulfone-protein A, the coupling of Tmab, and the evaluation in both HER2-overexpressing breast cancer cells (HCC1954) and non-overexpressing cells (MCF-7) by flow cytometry and fluorescence microscopy. Results show that the uptake is dependent on the level of overexpression of HER2, and the analysis of the cell viability reveals that targeted drugs are selective toward HCC1954, whereas MCF-7 cells remain unaffected.ConclusionProtein A-functionalized ISCOMs are versatile carriers that can be coupled to antibodies that act as targeting agents to deliver drugs. When coupling to Tmab and loading with paclitaxel or doxorubicin, they become efficient vehicles for the selective delivery of the drug to Tmab-resistant HER2-overexpressing breast cancer cells. These nanoparticles may pave the way for the development of novel therapies for poor prognosis resistant patients.

A new mechanism of trastuzumab resistance in gastric cancer: MACC1 promotes the Warburg effect via activation of the PI3K/AKT signaling pathway.

BackgroundTrastuzumab, a humanized antibody targeting HER2, exhibits remarkable therapeutic efficacy against HER2-positive gastric cancer. However, recurrent therapeutic resistance presents revolutionary claims. Warburg effect and AKT signaling pathway was involved in the resistance to trastuzumab. Our previous studies have demonstrated that overexpression of metastasis associated with the colon cancer 1 (MACC1) predicted poor prognosis of GC and promoted tumor cells proliferation and invasion. In this study, we found that MACC1 was significantly upregulated in trastuzumab-resistant cell lines. Besides, downregulation of MACC1 reversed this resistance.MethodsThe effect of trastuzumab and glycolysis inhibitor combination on cell viability, apoptosis, and cell metabolism was investigated in vitro using established trastuzumab-resistant GC cell lines. We assessed the impact of trastuzumab combined with oxamate on tumor growth and metabolism in an established xenograft model of HER2-positive GC cell lines.ResultsHere, we found that MACC1 was significantly upregulated in trastuzumab-resistant cell lines. Besides, downregulation of MACC1 in trastuzumab-resistant cells reversed this resistance. Overexpression of MACC1-induced trastuzumab resistance, enhanced the Warburg effect, and activated the PI3K/AKT signaling pathway, while downregulation of MACC1 presented the opposite effects. Moreover, when the PI3K/AKT signaling pathway was inhibited, the effects of MACC1 on resistance and glycolysis were diminished. Our findings indicated that MACC1 promoted the Warburg effect mainly through the PI3K/AKT signaling pathway, which further enhanced GC cells trastuzumab resistance.ConclusionsOur results indicate that co-targeting of HER2 and the Warburg effect reversed trastuzumab resistance in vitro and in vivo, suggesting that the combination might overcome trastuzumab resistance in MACC1-overexpressed, HER2-positive GC patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0302-1) contains supplementary material, which is available to authorized users.

An anti-ErbB2 fully human antibody circumvents trastuzumab resistance.

Trastuzumab, an anti-HER2/ErbB2 humanized antibody, has shown great clinical benefits in ErbB2-positive breast cancer treatment. Despite of its effectiveness, response rate to trastuzumab is limited and resistance is common. Here, we developed a new anti-ErbB2 antibody, denoted as H2-18, which was isolated from a phage display human antibody library. Previous studies have demonstrated that trastuzumab recognizes the juxtamembrane region of domain IV, and pertuzumab, another humanized ErbB2-specific antibody, binds to ErbB2 near the center of domain II. Our crystallographic analysis showed that the epitope recognized by H2-18 is within domain I of the ErbB2 molecule. H2-18 potently induced programmed cell death (PCD) in both trastuzumab-sensitive and -resistant breast cancer cell lines, while trastuzumab and pertuzumab, either used alone or in combination, only exhibits very weak PCD-inducing activity. More importantly, H2-18 could inhibit the growth of trastuzumab-resistant breast cancer cells far more effectively than trastuzumab plus pertuzumab, both in vitro and in vivo. In conclusion, H2-18 shows a unique ability to overcome trastuzumab resistance, suggesting that it has the great potential to be translated to the clinic.

HER2-Targeted Polyinosine/Polycytosine Therapy Inhibits Tumor Growth and Modulates the Tumor Immune Microenvironment.

The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer. Cancer Immunol Res; 4(8); 688-97. ©2016 AACR.

Abstract 4407: Inhibition of STAT3 in primary trastuzumab-resistant HER2-positive cells leads to decrease cell viability

Abstract Breast Cancer is the second leading cause of cancer related death in women. Approximately, 20%-30% of breast cancer patients overexpress Human Epidermal Growth Factor Receptor 2 (HER2). HER2 Is a Tyrosine Kinase receptor involved in proliferation and survival and its overexpression makes it a good target for patients with HER2 positive breast cancer. Trastuzumab (TRA) is a recombinant monoclonal antibody that binds HER2 receptor and inhibits its signaling. Unfortunately, clinical studies have demonstrated that 30% of patients that overexpress HER2 do not respond to Trastuzumab treatment. Therefore, understanding the molecular mechanisms underlying Trastuzumab resistance is essential to develop new therapies to treat these breast cancer patients. Previous studies in our lab demonstrated that a decrease in cell growth of HER2 overexpressing cells occurred when IGF2 and HER2 were inhibited. Nevertheless, these cells overcame the growth inhibition and the resulting proliferative growth coincided with an increase in STAT3 activation. STAT3 is a transcription factor that promotes cell proliferation and can regulate apoptosis in cancer cells. These findings lead us to hypothesize that blocking STAT3 together with IGF2 and HER2 will inhibit proliferation and cell viability of Trastuzumab resistant breast cancer cells. We treated Trastuzumab-resistant HER2 positive JIMT1 cells with HER2, IGF2, and/or STAT3 inhibitors. After 24 hours of treatment, there was a marked difference in cell morphology observed between the treatment groups. Cell viability was measured using WST-1, a reagent that measures mitochondrial activity. A significant decrease in cell viability was observed when cells were treated for 24 hours with a commercially available STAT3 inhibitor (Sttatic). There was no difference between the treatment groups when STAT3 was inhibited by Stattic or when STAT3 was inhibited in combination with IGF2 and/or HER2 inhibitors. These findings suggest that in Trastuzumab-resistant HER2 positive JIMT1 cells, STAT3 activation overcomes the inhibition of the HER2 and IGF2 proliferation pathways resulting in cell death prevention. Our study demonstrate that Trastuzumab-resistant HER2 positive JIMT1 cells can activate STAT3 as an alternative pathway to evade apoptosis and promote chemoresistance. Our data also illustrate the challenge of treating cancer based on single therapies since tumors have many alternative pathways to promote growth. Citation Format: Xousaen M. Helu, Alfida Fernandez, Daisy D. De Leon. Inhibition of STAT3 in primary trastuzumab-resistant HER2-positive cells leads to decrease cell viability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4407.

Abstract 2124: Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ breast cancer

Abstract ErbB-2 (HER2) is over expressed in 15 - 25% of breast cancer. First line therapy for HER2+ breast cancer is a humanized, monoclonal antibody, trastuzumab. Resistance is a major clinical problem. Previous work has suggested that Notch-1 is critical for resistance. PTEN, a breast tumor suppressor and potent inhibitor of the PI3-K/AKT is decreased in HER2+ breast cancer and loss of PTEN predicts for worst outcome. Notch-1 downregulates PTEN expression and we sought to determine the mechanism by which Notch-1 attenuates PTEN and whether this decrease is necessary for trastuzumab resistance. Our hypothesis is that Notch-1 decreases PTEN to maintain trastuzumab resistance. Human HER2+ breast cancer cell lines: sensitive (BT474HS) or resistant (BT474HR and HCC1954) to trastuzumab were used to measure Notch-1 modulation of PTEN expression when treated with trastuzumab. Protein and transcript expression of PTEN were measured by Western blot and RT-PCR, respectively. ChIP assays were preformed to identify whether Notch-1 regulated PTEN expression was mediated by direct recruitment of Notch-1 to the PTEN promoter. Growth and mammosphere forming assays were performed to determine if trastuzumab resistance could be rescued in trastuzumab treated cells with simultaneous Notch-1 and PTEN knockdown, or increased downstream PI3K pathway activity using a constitutive active form of AKT1 (Myr-AKT1). Western blot and RT-PCR results showed that Notch-1 protein levels are increased while PTEN mRNA and protein levels are decreased in trastuzumab resistant cells (BT-474HR) compared to sensitive (BT-474HS) cells. Results revealed that Notch-1 knockdown increased PTEN transcript and protein expression. Notch-1 ChIP assays demonstrated that Notch-1 was enriched on the PTEN promoter and a Notch target gene promoter, HEY1 in resistant cells. Notch1 knockdown significantly decreased proliferation and mammosphere forming efficiency (MFE) of resistant cells regardless of trastuzumab treatment. PTEN knockdown rescued the decrease in proliferation and MFE mediated by Notch-1 knockdown. As PTEN is an attenuator of PI3-K/AKT signaling, we asked whether constitutive activation of AKT1 was sufficient to rescue proliferation upon Notch-1 knockdown. The results showed that myr-AKT1 increased AKT1 signaling and rescued the decrease in proliferation mediated by Notch-1 knockdown. These results demonstrate, to our knowledge, for the first time that Notch-1 is a direct transcriptional repressor of PTEN in HER2+ breast cancer cells. Increased activation of the PI3K pathway through Notch-1 mediated inhibition of PTEN is necessary to maintain proliferation and possibly survival of the cancer stem cell population of trastuzumab resistant cells. Inhibiting the expression and/or activity of Notch-1 in HER2+ breast cancer could prove to be an effective therapy to prevent expansion of cancer stem cells and/or re-sensitize HER2+ breast cancer to ant-HER2 therapy. Citation Format: Andrew T. Baker, Kinnari Pandya, Clodia Osipo. Notch-1 mediated down regulation of PTEN in trastuzumab resistant HER2+ breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2124.

Abstract 4392: Yes1 is the key molecule for the resistance to trastuzumab in breast cancer, and dasatinib overcomes the resistance

Abstract Background: Overexpression of human epidermal growth factor receptor 2 (HER2) was observed in approximately 15-23% of breast cancers and they are classified as HER2-positive breast cancer. Trastuzumab is a therapeutic drug for the first choice for HER2-positive breast cancer, showing good response. However, acquired resistance to trastuzumab is one of the critical clinical issues in breast cancer treatment, especially in the patients with recurrent breast cancer. Therefore, it is necessary to develop the effective therapy to overcome the resistance. In this study, we established a trastuzumab-resistant breast cancer cell line from a trastuzumab-sensitive cell line with HER2 amplification (BT-474). We analyzed the mechanisms of resistance to trastuzumab and demonstrated the anti-tumor effect of dasatinib. Methods: Trastuzumab-resistant breast cancer cell line (BT-474-R) was established by treating BT-474 cells for long-term exposure with increasing doses of trastuzumab (from 0.1 μg/mL up to 40 μg/mL). Expression and activation of HER2 and its related molecules were investigated using western blotting and real-time PCR. Cell viability was evaluated using MTS assay. Cell cycle was analyzed using flow cytometry. Results: Proto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R. HER2 and Akt were also activated. Silencing of Yes1 by siRNA induced BT-474-R to recover sensitivity to trastuzumab. Pharmaceutical inhibition of Yes1 by Src inhibitor dasatinib was also effective to recovery sensitivity to trastuzumab in BT-474-R. Combination treatment of dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, these combination treatments induced G1-phase cell-cycle arrest and apoptosis. Conclusion: Yes1 plays an important role in acquired resistance to trastuzumab in HER2-positive breast cancer. Our data also suggest that pharmacological inhibition of Yes1 may become the new strategy to overcome resistance to trastuzumab. Citation Format: Hiromasa Yamamoto, Tatsuaki Takeda, Hirotaka Kanzaki, Ken Suzawa, Kei Namba, Hiroki Sato, Hidejiro Torigoe, Mototsugu Watanabe, Yuho Maki, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Yoshihisa Kitamura, Toshiaki Sendo, Shinichi Toyooka. Yes1 is the key molecule for the resistance to trastuzumab in breast cancer, and dasatinib overcomes the resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4392.

Abstract 2130A: Epigenetic silencing of TGFβ1, BCL6, KILLIN and CTSZ is related to trastuzumab resistance in HER2-positive breast cancer models

Abstract Introduction: The major clinical problem for HER2 breast cancer target therapies is the acquisition of resistance. Trastuzumab has been used for the treatment of HER2-positive early stage and metastatic breast cancers. Notoriously, up to 62% patients that respond to the initial treatment develop resistance within a year. Different cellular and molecular alterations may contribute to the development of Trastuzumab resistance, including epigenetic changes. The aim of our study has been the evaluation of epigenetic differences between Trastuzumab-sensitive and -resistant breast cancer cell lines to determine whether they might be used as potential biomarkers to help in the treatment assessment and monitoring. Experimental procedures: To understand the epigenetic changes that may be associated with Trastuzumab resistance we compared the DNA methylation status of ∼450000 CpG sites from a DNA methylation microarray performed in Trastuzumab-sensitive and resistant cells. To identify the differentially methylated CpGs we considered differences between methylation groups of ≥60%. The functions associated to genes were analysed using the Gene Ontology and were used to select a small number of candidate genes. Their differences in promoter methylation status were validated by expression techniques (qPCR) and subsequently confirmed by demethylation agent (5-aza-DC) treatment followed by qPCR. The Trastuzumab resistant HER2-positive cell line (SKTR) had been previously developed in our laboratory by exposing SKBr3 cells to Trastuzumab, starting with 1μM for three months of exposure and increasing up to 2 μM for a 12 months period. Summary of the new, unpublished data: The DNA methylation microarray data showed that different 5’-CpG island sites had altered methylation profile when comparing Trastuzumab-resistant (SKTR) and -sensible SKBr3 cells. We selected 4 candidate genes with a hypermethylation profile in -resistant vs. -sensible cells: TGFβ1 (Transforming Growth Factor Beta 1), BCL6 (B-Cell CLL/Lymphoma 6), KILLIN (P53-Regulated DNA Replication Inhibitor) and CTSZ (Cathepsin Z). The hypermethylation of these four candidate genes in SKTR was in agreement with their expression downregulation observed by PCR analysis. SKTR treatment with the demethylation agent 5-aza-2’-deoxycytidine restored the expression levels of all four genes to the levels of Trastuzumab-sensitive cells. Although previously related to breast cancer, TGFβ1, BCL6, KILLIN and CTSZ have not earlier been associated with the development of Trastuzumab resistance in breast cancer or in other cancer types and may be further studied as potential biomarkers. Conclusions: These results provide a basis for future studies to validate the hypermethylation status of these genes as a predictive or monitoring biomarker of Trastuzumab resistance in HER2+ breast cancer patients. Citation Format: Sonia Palomeras, Angel Diaz-Lagares, Sarrats Ariadna, Crujeiras Ana Belen, Giro-Perafita Ariadna, Juan Sandoval, Marc Rabionet, Manel Esteller, Teresa Puig. Epigenetic silencing of TGFβ1, BCL6, KILLIN and CTSZ is related to trastuzumab resistance in HER2-positive breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2130A.

Anti-HER3 Monoclonal Antibody Inhibits Acquired Trastuzumab-Resistant Gynecologic Cancers.

Antibody resistance, both de novo and acquired, is usually related to high risk of recurrence and lower survival rate in gynecologic cancers. Prevention or reversal of the resistance often yields beneficial clinical results. It was reported that anti-human epidermal growth factor receptor 3 monoclonal antibody was effective against trastuzumab-resistant breast cancer cells. Here in our laboratory, an acquired trastuzumab-resistant ovarian cancer cell line, SKOV3-T, was established previously. Further, human epidermal growth factor receptor 3 was observed to be upregulated in this cell line by microarray detection, suggesting that the antagonist against human epidermal growth factor receptor 3 might be effective to inhibit the resistant cells. We developed an anti-human epidermal growth factor receptor 3 monoclonal antibody, LMAb3, and its affinity to bind human epidermal growth factor receptor 3 was calculated by the Biacore method. Preliminarily, LMAb3's antitumor activity was evaluated in vitro using cell growth/proliferation and clone formation assays in the breast cancer cell line MCF-7. Furthermore, LMAb3 was also evaluated for its inhibitory effect on the carcinogenicity of the SKOV3-T cells, which were induced to overexpress human epidermal growth factor receptor 3, both in vitro and in vivo. The possible underlying signal transduction mechanisms were also identified by Western blot in the MCF-7 and SKOV3-T cells. LMAb3 was able to inhibit the cell growth/proliferation, clone, and tumor formation both in vitro (in the MCF-7 and SKOV3-T cells) and in vivo. The underlying mechanism of LMAb3 possibly involves inactivation of the HER family proteins (human epidermal growth factor receptor 1, human epidermal growth factor receptor 2, and especially human epidermal growth factor receptor 3) as well as the downstream mitogen-activated protein kinase and protein kinase B pathways. Our work suggests that satisfactory curative effects might be achieved with LMAb3 to treat the trastuzumab-resistant, human epidermal growth factor receptor 3-positive cases of gynecologic cancers.

571 - Identification of molecular mechanisms of acquired resistance to trastuzumab in gastric cancer

The HER2 receptor is overexpressed in 7-34% of gastric cancer (GC) tumors, being a potential therapeutic target. Trastuzumab, an anti-HER2 monoclonal antibody, in combination with platinum therapy was approved in 2010 as first-line treatment for advanced GC tumors overexpressing HER2. Despite the clinical benefit of trastuzumab, development of acquired resistance to treatment limits the efficacy of trastuzumab. To study the mechanisms of trastuzumab acquired resistance we generated, upon chronic exposure to trastuzumab, a trastuzumab-resistant cell line derived from the human HER2-positive GC cell NCI-N87, hereafter named NTR2.4. The resistant phenotype of NTR2.4 cell line was associated with an increased basal activation of SRC, MAPK/ERK and PI3K/mTOR HER2 downstream pathways that persisted upon exposure to trastuzumab. Trastuzumab treatment induced a compensatory upregulation of HER3 and an increased expression of the HER family ligands EGF, AREG, HB-EGF, and NRG1 in NTR2.4 compared with the trastuzumab-sensitive NCI-N87 cell line. Ligand stimulation induced resistance to trastuzumab in NCI-N87 cells. These results were replicated in another model of trastuzumab-resistance, OTR6 cell line, suggesting an important role of HER receptor plasticity and HER family ligand in trastuzumab efficacy and resistance. Analysis of clinical samples from HER2-positive GC patients treated with trastuzumab showed higher EGF concentration in serum samples and increased EGF mRNA expression in tumor biopsies at trastuzumab progression compared with samples previous to trastuzumab treatment. Treatment with Pan-HER, a novel antibody mixture against EGFR, HER2, and HER3, was effective in both trastuzumab-sensitive and trastuzumab-resistant cell lines and also in the presence of ligands. Altogether, our results suggested that Pan-HER would be a potential candidate to overcome trastuzumab resistance in advanced GC patients.

The voltage gated Ca(2+)-channel Cav3.2 and therapeutic responses in breast cancer.

BackgroundUnderstanding the cause of therapeutic resistance and identifying new biomarkers in breast cancer to predict therapeutic responses will help optimise patient care. Calcium (Ca2+)-signalling is important in a variety of processes associated with tumour progression, including breast cancer cell migration and proliferation. Ca2+-signalling is also linked to the acquisition of multidrug resistance. This study aimed to assess the expression level of proteins involved in Ca2+-signalling in an in vitro model of trastuzumab-resistance and to assess the ability of identified targets to reverse resistance and/or act as potential biomarkers for prognosis or therapy outcome.MethodsExpression levels of a panel of Ca2+-pumps, channels and channel regulators were assessed using RT-qPCR in resistant and sensitive age-matched SKBR3 breast cancer cells, established through continuous culture in the absence or presence of trastuzumab. The role of Cav3.2 in the acquisition of trastuzumab-resistance was assessed through pharmacological inhibition and induced overexpression. Levels of Cav3.2 were assessed in a panel of non-malignant and malignant breast cell lines using RT-qPCR and in patient samples representing different molecular subtypes (PAM50 cohort). Patient survival was also assessed in samples stratified by Cav3.2 expression (METABRIC and KM-Plotter cohort).ResultsIncreased mRNA of Cav3.2 was a feature of both acquired and intrinsic trastuzumab-resistant SKBR3 cells. However, pharmacological inhibition of Cav3.2 did not restore trastuzumab-sensitivity nor did Cav3.2 overexpression induce the expression of markers associated with resistance, suggesting that Cav3.2 is not a driver of trastuzumab-resistance. Cav3.2 levels were significantly higher in luminal A, luminal B and HER2-enriched subtypes compared to the basal subtype. High levels of Cav3.2 were associated with poor outcome in patients with oestrogen receptor positive (ER+) breast cancers, whereas Cav3.2 levels were correlated positively with patient survival after chemotherapy in patients with HER2-positive breast cancers.ConclusionOur study identified elevated levels of Cav3.2 in trastuzumab-resistant SKBR3 cell lines. Although not a regulator of trastuzumab-resistance in HER2-positive breast cancer cells, Cav3.2 may be a potential differential biomarker for survival and treatment response in specific breast cancer subtypes. These studies add to the complex and diverse role of Ca2+-signalling in breast cancer progression and treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0299-0) contains supplementary material, which is available to authorized users.