Trastuzumab-resistant Cells Research Articles

Abstract P4-03-15: Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer

Abstract Background: Afatinib is an irreversible pan-HER inhibitor approved for non-small cell lung cancer. We have previously shown that afatinib inhibits growth of HER2-positive breast cancer cells and enhances response to trastuzumab. However, we have also shown that long-term exposure to tyrosine kinase inhibitors leads to the development of acquired resistance. To determine if acquired afatinib resistance develops in HER2-positive breast cancer cells, we exposed a HER2-positive breast cancer cell line to afatinib for 6 months and investigated alterations in the cells following long-term exposure. Methods: SKBR3 cells were treated with 150 nM afatinib twice-weekly for 6 months. Growth response to drug inhibitors was assessed by acid phosphatase assay. Drug sensitivity was examined in four HER2-positive cell lines (SKBR3, EFM192A, BT474 and HCC1954) and three acquired trastuzumab resistant cell lines (SKBR3-T, BT474-T and EFM192A-T). Reverse phase protein array (RPPA) was used to determine alterations in key signaling pathways. Src, p-Src, EGFR, p-EGFR, ERK1/2, p-ERK 1/2 levels were examined by Western blotting. To examine the prevention of the development of afatinib resistance, cells were treated twice weekly with afatinib, dasatinib, or the combination and stained with crystal violet when confluent. Results: Following 6 months of afatinib treatment, the SKBR3-A cells were more resistant to afatinib compared to parental cells (IC50 SKBR3-A 284 ± 28.2 nM vs SKBR3-Par 10.9 ± 3.4 nM). Furthermore, the resistant cells were cross-resistant to lapatinib, neratinib and trastuzumab. RPPA interrogation of the SKBR3-A cells showed alterations in several pathways, including significantly increased levels of p-Src (Y416). SKBR3-A cells were more sensitive to Src inhibition with dasatinib compared to SKBR3-Par cells and the combination of afatinib and dasatinib was highly synergistic in SKBR3-A cells (CI value = 0.09 ± 0.06). The combination of afatinib and dasatinib was also synergistic in the trastuzumab resistant SKBR3-T cells (Table). Afatinib and dasatinib inhibited EGFR and Src activation and ERK 1/2 signalling in SKBR3-A cells. Short-term resistance assays showed that the addition of dasatinib to afatinib blocks the emergence of resistant cells in three of four HER2 positive cell lines tested and two of the three acquired trastuzumab resistant cell lines tetsed. Conclusion: HER2-positive breast cancer cells that are highly sensitive to afatinib can develop acquired resistance to afatinib within six months. Src is a potential target to prevent the development of afatinib resistance and thus combined treatment with afatinib and dasatinib may be beneficial in patients with HER2-positive breast cancer. Percentage growth in HER2-positive cells treated with afatinib and/or dasatinib for 5 days. 20 nM Afatinib40 nM Dasatinib20 nM Afatinib + 40 nM DasatinibSKBR325.2 ± 6.1109.9 ± 6.222.6 ± 6.2SKBR3-A62.1 ± 0.787.3 ± 11.036.3 ± 0.3*SKBR3-T42.6 ± 4.9107.4 ± 9.729.3 ± 3.5*BT47418.2 ± 3.079.5 ± 5.213.3 ± 1.1BT474-T6.9 ± 2.795.3 ± 2.06.2 ± 2.3EFM192A39.1 ± 4.7105.7 ± 3.135.3 ± 2.7EFM192A-T30.4 ± 5.299.8 ± 2.528.5 ± 4.4HCC195461.8 ± 8.180.5 ± 8.219.4 ± 3.6** indicates enhanced anti-proliferative response for the combination compared to the single agents. Citation Format: Conlon N, Canonici A, Morgan C, Cremona M, Hennessey BT, Eustace A, O'Brien N, Slamon D, Crown J, O'Donovan N. Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-15.

Abstract OT3-06-05: A phase Ib/II trial of coPANlisib in combination with tratuzumab in pretreated recurrent or metastatic HER2-positive breast cancer “PantHER”

Abstract Background The phosphoinositide 3 kinase (PI3K) pathway is important in the oncogenic function of HER2. Aberrent activation of PI3K is implicated in resistance to trastuzumab and other HER2-targeted therapies and is frequent, with up to 22% of HER2 positive breast cancer having a PIK3CA mutation. Copanlisib is a pan-class 1 PI3K inhibitor that shows particular activity against PI3Kα, the isoform encoded by the PIK3CA gene. Copanlisib has been shown to re-sensitise trastuzumab resistant cell lines to trastuzumab with synergism seen in some cell lines between copanlisib and HER2 targeted therapy. Trial design The study is a phase Ib/II open label, single arm adaptive, multi-centre trial of copanlisib in combination with trastuzumab. Eligible patients are treated with a dose escalation schedule of copanlisib IV on Days 1, 8 and 15 of a 28 day cycle with trastuzumab 2 mg/kg weekly (loading dose of 4 mg/kg in cycle 1). The phase II dose will be based on the maximum tolerated dose (MTD) established in Phase Ib. Patients are treated until radiologic or symptomatic progression, unacceptable toxicity, consent withdrawal or physician's decision. Eligibility criteria Eligible patients must have recurrent incurable or metastatic HER2-positive breast cancer that has progressed on at least one prior line of trastuzumab or T-DM1-based treatment regimen in this setting. Patients with treated and controlled brain metastases are eligible. Participants must have adequate organ function and ECOG PS ≤ 2. Patients recruited for the Phase II part of the study must have a PIK3CA mutation. Patients with uncontrolled arterial hypertension, uncontrolled diabetes or recent clinically serious infections are excluded. Specific aims The primary end point for the phase Ib part of this study is to determine the MTD for the combination. For the phase II study is anti-tumour efficacy, measured by Clinical Benefit Rate (CBR). Secondary end points are evaluation of safety and tolerability, progression-free survival, time to treatment failure, duration of response and overall survival. Incorporated translational endpoints include examination of molecular tumor adaptation in tissue and blood. Given the role of PI3K in cellular glucose metabolism, an additional exploratory objective is to determine if quantitive reduction in metabolic signal on Positron Emission Tomography-Computed Tomography (PET-CT) is predictive of benefit from therapy. Statistical methods To establish the MTD, we use a modified 3+3 design where 3 additional patients will be accrued even if the first 3 patients accrued experience no dose limiting toxicities (DLT) in sequential cohorts for a planned 12 patients. To determine the CBR, a one sample exact binomial test with a one sided significance level of 5%, 19 evaluable patients will provide >80% power to detect a difference between the null hypothesis proportion of 30% for CBR versus the alternative hypothesis proportion of 65%. Present accrual and target accrual There are 9 patients recruited so far to the phase Ib part of this study. Target accrual is 12 and for phase II is 19 patients. Contact information for people with a specific interest in the trial Prof Bryan Hennessy, Beaumont Hospital, Dublin Ireland Funded by Bayer Citation Format: Keegan NM, Walshe J, Gullo G, Kennedy J, Bulger K, Kelly CM, Crown J, Toomey S, Egan K, Kerr J, Given M, Hernando A, Teiserskiene A, Grogan L, Breathnach O, Morris PG, Keane M, Hennessy BT. A phase Ib/II trial of coPANlisib in combination with tratuzumab in pretreated recurrent or metastatic HER2-positive breast cancer “PantHER” [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-06-05.

The HSP90 inhibitor NVP-AUY922 inhibits growth of HER2 positive and trastuzumab-resistant breast cancer cells

As HER2 is a client protein of the molecular chaperone Hsp90, targeting Hsp90 may be beneficial in HER2-positive breast cancer. In this study, the activity of the Hsp90 inhibitor NVP-AUY922 was assessed in HER2 overexpressing breast cancer cell lines, including two cell line models of acquired trastuzumab-resistance. The seven HER2-positive breast cancer cell lines tested showed significant sensitivity to NVP-AUY922 in vitro, with IC50 values between 6 and 17nM. Combining NVP-AUY922 with chemotherapy did not improve response. NVP-AUY922 in combination with trastuzumab, significantly enhanced growth inhibition in three of the seven cell lines tested. In conclusion, our data shows that NVP-AUY922 displays potent anti-cancer activity in HER2-positive and trastuzumab-resistant breast cancer cells, and supports further testing of NVP-AUY922 in patients with HER2-positive breast cancer.

MiR-200c inhibits TGF-β-induced-EMT to restore trastuzumab sensitivity by targeting ZEB1 and ZEB2 in gastric cancer.

Gastric cancer is the fifth most common malignancy in the world, with Eastern Asia as one of areas with the highest incidence rates. Trastuzumab, a HER2-targeting antibody, combined with chemotherapy has been successfully employed for the gastric cancer patients with HER2 overexpression/amplification. However, trastuzumab resistance is a major problem in clinical practice. Here we observed that the trastuzumab-resistant gastric cancer cell line NCI-N87/TR expressed high levels of epithelial-mesenchymal transition factors and demonstrated increased migration and invasion capability compared with NCI-N87 cells. Downregulated E-cadherin and increased N-cadherin, TGF-β, ZEB1, ZEB2, TWIST1, and Snail were detected in NCI-N87/TR cells. We also found that miR-200c was downregulated in NCI-N87/TR cells compared with parental cells NCI-87 by qRT-PCR. Treatment with TGF-β downregulated the expression of miR-200c and upregulated ZEB2, and significantly decreased the trastuzumab sensitivity of NCI-N87 cells. miR-200c restored trastuzumab sensitivity and inhibited migration and invasion through suppressing ZEB1 and ZEB2. In summary, TGF-β/ZEB2 axis plays an encouraging role in trastuzumab resistance of gastric cancer, while miR-200c overexpression downregulates ZEB1/ZEB2 and resensitizes drugs resistance. Our findings might provide a potential therapeutic strategy for trastuzumab resistance of gastric cancer.

SHP-1 reverses trastuzumab resistance and migration in HER2-positive breast cancer cells through the stat3/p-stat3 pathway

Decreased expression of Src homology region 2 domain containing protein tyrosine phosphatase 1 (SHP-1) was found in human breast cancer cell lines and primary breast cancers. As a candidate tumor suppressor gene, the role of SHP-1 in HER-2-positive breast cancer remains to be elucidated. The aim of the present study was to investigate the effects of upregulated SHP-1 expression in reversing trastuzumab resistance and migration by HER-2-positive breast cancer cells, potentially providing a new target to trastuzumab resistance in anti-HER2 therapy. Western blotting analysis was used to determine the expression levels of SHP-1 in trastuzumab-resistant cells transfected with a lentivirus vector expressing GFP-(control) or GFP-SHP-1 (experimental). MTT assay was used to determine changes in sensitivity to trastuzumab between two modified cell lines after overexpressing SHP-1. Clone formation and wound-healing assays were used to detect the capacity change in cell proliferation and migration. Western blot analysis was also used to examine the expressions of c-myc, cyclinD1, p-STAT3, STAT3, and MMP-9. Results from this study indicated that SHP-1 not only reduced cell proliferation and migration, but also significantly sensitized resistant cells to trastuzumab. Data also showed that SHP-1 restores sensitivity to trastuzumab in drug-resistant cell lines. Mechanistic analyses indicated that SHP-1 might target STAT3 to inactivate p-STAT3 and reduce expression of its downstream signaling pathway. In conclusion, results presented here suggest that targeting SHP-1 may represent a potential approach to re-sensitize previously resistant cells to trastuzumab treatment.

SP1 promotes tumor angiogenesis and invasion by activating VEGF expression in an acquired trastuzumab‑resistant ovarian cancer model.

Ovarian cancer is one of the most common gynecologic cancers and the leading cause of mortality in women worldwide. HER2/neu is overexpressed in various types of cancers and is most commonly associated with decreased survival. Trastuzumab is a humanized anti-HER2 monoclonal antibody for the treatment of HER2-positive breast cancers. However, primary and/or acquired resistance occurs in up to 62% patients during the first year of treatment. Vascular endothelial growth factor (VEGF) is a well-known angiogenesis factor involved in many physiological and pathological processes. Its significance has been implicated in promoting tumor growth and metastasis via angiogenesis. In the present study, we demonstrated that the upregulation of SP1 enhanced expression of VEGF promoting the angiogenesis and migration of trastuzumab-resistant ovarian cancer cell line SKOV3-T. Our in vitro and in vivo results both gave evidence that the SP1-VEGF axis was responsible for the enhanced malignancy, angiogenesis and migration in the acquired trastuzumab-resistant ovarian cancer cell model.

DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer.

The majority of patients develop resistance against suppression of HER2-signaling mediated by trastuzumab in HER2 positive breast cancer (BC). HER2 overexpression activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. MAPK phosphatases (MKPs) are essential regulators of MAPKs and participate in many facets of cellular regulation, including proliferation and apoptosis. We aimed to identify whether differential MKPs are associated with resistance to targeted therapy in patients previously treated with trastuzumab. Using gene chip data of 88 HER2-positive, trastuzumab treated BC patients, candidate MKPs were identified by Receiver Operator Characteristics analysis performed in R. Genes were ranked using their achieved area under the curve (AUC) values and were further restricted to markers significantly associated with worse survival. Functional significance of the two strongest predictive markers was evaluated in vitro by gene silencing in HER2 overexpressing, trastuzumab resistant BC cell lines SKTR and JIMT-1. The strongest predictive MKPs were DUSP4/MKP-2 (AUC=0.75, p=0.0096) and DUSP6/MKP-3 (AUC=0.77, p=5.29E-05). Higher expression for these correlated to worse survival (DUSP4: HR=2.05, p=0.009 and DUSP6: HR=2, p=0.0015). Silencing of DUSP4 had significant sensitization effects – viability of DUSP4 siRNA transfected, trastuzumab treated cells decreased significantly compared to scramble-siRNA transfected controls (SKTR: p=0.016; JIMT-1: p=0.016). In contrast, simultaneous treatment with DUSP6 siRNA and trastuzumab did not alter cell proliferation. Our findings suggest that DUSP4 may represent a new potential target to overcome trastuzumab resistance.

A small-molecule inhibitor of SMAD3 attenuates resistance to anti-HER2 drugs in HER2-positive breast cancer cells.

Resistance against anti-HER2 drugs in HER2-positive breast cancer is a major obstacle to the improving prognosis. Transforming growth factor β (TGFβ) is a cytokine involved in the acquisition of more malignant phenotypes through epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. The aim of this study was to investigate the effects of TGFβ and its downstream SMAD pathway on resistance to anti-HER2 drugs. HER2-positive breast cancer cell lines were stimulated with TGFβ for 14days. Then, the sensitivity to trastuzumab and lapatinib and the expression levels of various EMT and CSC markers were examined. The correlation of nuclear SMAD3 expression in untreated breast tumor tissues with trastuzumab efficacy in neoadjuvant settings was examined. The effect of a small-molecule inhibitor of SMAD3 (SIS3) on resistance to anti-HER2 drugs was explored. We found that continuous activation of the TGFβ-SMAD3 pathway induced resistance to anti-HER2 drugs and CSC traits in HER2-positive breast cancer cells. The induction of drug resistance by TGFβ required strong activation of SMAD3. In fact, activated SMAD3 regulated multiple genes that harbor SMAD-binding elements and are involved in trastuzumab resistance. Nuclear SMAD3 expression in tumor tissue was inversely correlated with sensitivity to neoadjuvant treatment with trastuzumab. SIS3 not only prevented the acquisition of resistance to anti-HER2 drugs but also restored trastuzumab sensitivity in trastuzumab-resistant cells. This study indicates that the TGFβ-SMAD3 pathway plays an important role in the induction and maintenance of resistance to anti-HER2 drugs. Thus, SMAD3 is a potential therapeutic target that can inhibit resistance and restore sensitivity to anti-HER2 drugs.

Abstract 115: Flubendazole targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer with trastuzumab resistance

Abstract Background and Propose: HER2-positive breast cancers are associated with an aggressive phenotype and trastuzumab resistance leading to poor clinical outcomes. Flubendazole (FLU) is a potent anthelmintic agent that inhibits microtubule polymerization, and also exhibits anticancer activity in several cancer types. The objective of the present study was to investigate the effect of FLU on apoptosis, HER2/Akt signaling pathway, cancer stem cell (CSC)-like properties and trastuzumab-resistance in HER2-positive breast cancer cells. Methods: FLU-induced antitumor effects were characterized by MTS assay, Sub-G1 and G2/M-phase arrest population, Annexin-V analysis, ALDH1 activity assay, Western blotting and immunofluorescence. For in vivo study, trastuzumab-resistant JIMT1 cells were injected into the mammary fat pads of BALB/c nude mice. We then determined the tumor growth and CSC-like properties. Results and conclusion: FLU significantly reduced cell viability and induced apoptosis with accompanied by activation of caspase-3, -7, and -8, as well as PARP cleavage in HER2-positive cell lines (MDA-MB-453, SKBR3 and BT474). FLU-induced apoptosis is associated with significant decreases in expression levels of HER2, p-HER2 (Tyr1221/1222), HER3, and p-HER3 (Tyr1289). FLU treatment also resulted in G2/M-phase arrest of the cell cycle, as evidenced by a marked downregulation of phospho-Histone H3 expression. These results were closely related to the decline of CSC-like properties, coinciding with the suppression of ALDH1 activity. These effects were also observed in trastuzumab-resistant JIMT-1 cells, and significant reductions in tumor growth and ALDH1A1 downregulation were observed in xenograft models together with downregulation of HER2 expression in vivo. Our findings suggest that FLU may be potentially effective for the treatment of HER2-positive breast cancer with trastuzumab resistance. Note: This abstract was not presented at the meeting. Citation Format: Daeil Sung, Youngkwan Cho, Eunhye Oh, Tae-Min Cho, Yoon-Jae Kim, Ji Young Kim, Jae hong Seo. Flubendazole targets cancer stem-like properties and the HER2/Akt signaling pathway in HER2-positive breast cancer with trastuzumab resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 115. doi:10.1158/1538-7445.AM2017-115

Abstract 5869: Resistance mechanism against trastuzumab in HER2-positive cancer cells and its negation by Src inhibition

Abstract Background: Trastuzumab in combination with chemotherapy is a standard of care for patients with HER2-positive breast and gastric cancer. Resistance mechanism to trastuzumab, anti-HER2 therapy, includes multiple pathways. Among them, Src is not well known especially in HER2-positive gastric and biliary tract cancers. We investigated the role of Src involved in trastuzumab resistance and explored the potential of Src inhibition as a trastuzumab resistance overcoming strategy. Methods: Four trastuzumab-resistant (HR) cells (SNU216HR, N87HR, SNU2670HR, SNU2773HR) were established from 2 HER2-amplified gastric cancer cells (SNU216, NCI-N87) and 2 HER2-amplified biliary tract cancer cells (SNU2670, SNU2773). For Src inhibition, bosutinib was used. MTT assay, colony formation assay, cell cycle analysis by FACS Calibur flow cytometer, and cell migration assay were done. Animal experiments were conducted to test anti-tumor effect of bosutinib using SNU2670 and SNU2670HR xenograft models. Results: SNU2670HR/NCI-N87HR cells showed pSrc activation, in contrast, SNU216HR/SNU2773HR cells exhibited decreased pSrc expression. In these pSrc decreased HR cells, pFAK was elevated. Bosutinib downregulated Src-FAK signaling more obviously in Src activated HR cells than parental cells. In pSrc decreased HR cells, bosutinib reduced Src-dependent FAK phosphorylation to affect cell fate. Bosutinib inhibited the growth of both parental cells and HR cells, and induced apoptosis and G1 arrest in HR cells. Bosutinib suppressed HR cell migration more effectively compared with parental cells. Bosutinib exhibited potent tumor growth inhibition in both SNU2670 and SNU2670HR xenograft models and more significantly suppressed tumor growth in HR models. Conclusion: Src activation may contribute to trastuzumab resistance in part in HER2-positive gastric and biliary tract cancer cells. Targeting Src might be a candidate strategy to overcome trastuzumab resistance in HER2-positive cancers. Citation Format: Meihua Jin, Ah Rong Nam, Ji Eun Park, Ju Hee Bang, Yung Jue Bang, Do Youn Oh. Resistance mechanism against trastuzumab in HER2-positive cancer cells and its negation by Src inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5869. doi:10.1158/1538-7445.AM2017-5869

Release of HER2 repression of trefoil factor 3 (TFF3) expression mediates trastuzumab resistance in HER2+/ER+ mammary carcinoma.

HER2+/ER+ breast cancer, a subset of the luminal B subtype, makes up approximately 10% of all breast cancers. The bidirectional crosstalk between HER2 and estrogen receptor (ER) in HER2+/ER+ breast cancer contributes to resistance towards both anti-estrogens and HER2-targeted therapies. TFF3 promotes breast cancer progression and has been implicated in anti-estrogen resistance in breast cancer. Herein, we investigated the cross-regulation between HER2 and estrogen-responsive TFF3, and the role of TFF3 in mediating trastuzumab resistance in HER2+/ER+ breast cancer. TFF3 expression was decreased by HER2 activation, and increased by inhibition of HER2 with trastuzumab in HER2+/ER+ breast cancer cells, partially in an ERα-independent manner. In contrast, the forced expression of TFF3 activated the entire HER family of receptor tyrosine kinases (HER1-4). Hence, HER2 negatively regulates its own signalling through the transcriptional repression of TFF3, while trastuzumab inhibition of HER2 results in increased TFF3 expression to compensate for the loss of HER2 signalling. In HER2+/ER+ breast cancer cells with acquired trastuzumab resistance, TFF3 expression was markedly upregulated and associated with a corresponding decrease in HER signalling. siRNA mediated depletion or small molecule inhibition of TFF3 decreased the survival and growth advantage of the trastuzumab resistant cells without re-sensitization to trastuzumab. Furthermore, TFF3 inhibition abrogated the enhanced cancer stem cell-like behaviour in trastuzumab resistant HER2+/ER+ breast cancer cells. Collectively, TFF3 may function as a potential biomarker and therapeutic target in trastuzumab resistant HER2+/ER+ breast cancer.

Resistance Mechanism against Trastuzumab in HER2-Positive Cancer Cells and Its Negation by Src Inhibition.

Trastuzumab in combination with chemotherapy is the standard of care for patients with human epidermal growth factor receptor 2 (HER2)-positive breast and gastric cancers. Several resistance mechanisms against anti-HER2 therapy have been proposed. Src activation has been suggested to be responsible for the resistance of HER2-positive breast cancer. In our study, we generated four trastuzumab-resistant (HR) cancer cell lines from HER2-amplified gastric and biliary tract cancer cell lines (SNU-216, NCI-N87, SNU-2670, and SNU-2773). Elevated Src phosphorylation was detected in SNU2670HR and NCI-N87HR cell lines, but not in SNU216HR or SNU2773HR cell lines. In SNU216HR and SNU2773HR cell lines, phospho-FAK (focal adhesion kinase) was elevated. Bosutinib as a Src inhibitor suppressed growth, cell-cycle progression, and migration in both parental and HR cell lines. Specifically, Src interacted with FAK to affect downstream molecules such as AKT, ERK, and STAT3. Bosutinib showed more potent antitumor effects in Src-activated HR cell lines than parental cell lines. Taken together, this study suggests that Src inhibition may be an effective measure to overcome trastuzumab resistance in HER2-positive cancer. Mol Cancer Ther; 16(6); 1145-54. ©2017 AACR.

A phase I study of the bispecific antibody T-cell engager GBR 1302 in subjects with HER2-positive cancers.

TPS3091 Background: GBR 1302, a bispecific antibody based on Glenmark’s BEAT platform, is designed to recruit cytotoxic T-cells (independent of their specificity) to HER2-positive cancer cells where they are activated by the CD3e-specific domain of the molecule. Preclinically, GBR 1302 has demonstrated potent killing of HER2-positive human cancer cells (HER2 3+ or 2+ by IHC HercepTest), as well as growth suppression of the trastuzumab-resistant cell line JIMT-1. In contrast, the GBR 1302 concentration required to kill primary cardiomyocytes with normal HER2 levels was up to 1000 times greater than the concentration needed to kill HER2 3+ tumor cell lines. This study will determine safety and tolerability of GBR 1302 monotherapy in subjects with HER2-positive cancers. Methods: Part 1 (dose-finding) of this ongoing phase 1 study (NCT02829372) is enrolling adults with progressing HER2-positive solid tumors for which no standard treatment is available. Intravenous GBR 1302 is given every 2 weeks in 28-day cycles at escalating doses (Table). Each of the first 4 cohorts includes a single subject; subsequent cohorts enroll subjects using a standard 3+3 design. Primary endpoints are: maximum tolerated dose (MTD) of GBR 1302; and relationship of GBR 1302 with the incidence, nature, and intensity of adverse events. After Cycle 1, subjects continue GBR 1302 treatment until disease progression or unacceptable toxicity. Part 2 (expansion) will treat subjects at the MTD to further evaluate anti-tumor activity, as well as safety and pharmacokinetics. Due to the known cardiotoxic potential of classic HER2-targeting strategies, this study incorporates a rigorous serological and echocardiographic surveillance schedule. The effects of GBR 1302 on the adaptive immune system will also be studied at cellular and serological levels in translational research. Clinical trial information: NCT02829372. [Table: see text]

The anti-ErbB2 antibody H2-18 and the pan-PI3K inhibitor GDC-0941 effectively inhibit trastuzumab-resistant ErbB2-overexpressing breast cancer.

Trastuzumab, an anti-ErbB2 humanized antibody, brings benefit to patients with ErbB2-amplified metastatic breast cancers. However, the resistance to trastuzumab is common. Our previously reported H2-18, an anti-ErbB2 antibody, potently induced programmed cell death in trastuzumab-resistant breast cancer cells. Here, we aim to investigate the antitumor efficacy of H2-18 in combination with the pan-PI3K inhibitor GDC-0941 in trastuzumab-resistant breast cancer cell lines. The results showed that H2-18 and GDC-0941 synergistically inhibited the in vitro proliferation of BT-474, SKBR-3, HCC-1954 and HCC-1419 breast cancer cells. H2-18 plus GDC-0941 showed significantly enhanced programmed cell death-inducing activity compared with each drug used alone. The combination of H2-18 and GDC-0941 did not increase the effect of single agent on ROS production, cell cycle and ErbB2 signaling. Importantly, the in vivo antitumor efficacy of H2-18 plus GDC-0941 was superior to that of single agent. Thus, the enhanced in vivo antitumor efficacy of H2-18 plus GDC-0941 may mainly be attributable to its increased programmed cell death-inducing activity. Collectively, H2-18 plus GDC-0941 could effectively inhibit tumor growth, suggesting the potential to be translated into clinic as an efficient strategy for ErbB2-overexpressing breast cancers.

Activation of STAT3/HIF-1α/Hes-1 axis promotes trastuzumab resistance in HER2-overexpressing breast cancer cells via down-regulation of PTEN

BackgroundResistance to the HER2-targeted antibody trastuzumab remains to be a major clinical challenge in the treatment of HER2-positive breast cancer. Hyper-activation of STAT3 is proposed to be a predictive biomarker of trastuzumab resistance. However, the precise mechanism(s) remains poorly defined. Evidence is emerging that HIF-1α, a central downstream element of STAT3 pathway, serves a pivotal role in the complex signaling network with subsequent diverse cellular events. Material and methodsWe have established trastuzumab resistant SKBR3 cells (SKBR3-TR). The cell viability, apoptosis as well as western blot, siRNA transfection and co-immunoprecipitation assays were performed to evaluate the involvement of STAT3/HIF-1α in modulation of trastuzumab resistance. ResultsWe found that in SKBR3-TR cells and conditioned medium-treated parental cells, constitutive phosphorylated STAT3 coincided with prominent up-regulation of HIF-1α which was accompanied with PTEN attenuation. Moreover, the inhibition of STAT3 activation by Stattic and/or genetically STAT3 knocking down decreased HIF-1α level in SKBR3-TR cells. Additionally, treatment with Stattic and/or STAT3 siRNA engendered the up-regulation of PTEN protein in STAT3-inhibited resistant cells. Restoration of PTEN was also observed following siRNA-mediated silencing of HIF-1α expression. Moreover, down-regulation of HIF-1α caused a reduction in the HES-1 content. Further study with HES-1 specific siRNA revealed the elevation of PTEN expression in HES-1 knock-down trastuzumab resistant cells. ConclusionThe impairment of STAT3-HIF-1α-HES-1 pathway restored trastuzumab sensitivity through up-regulation of PTEN protein. General significanceThese findings highlighted the signal integrator role of HIF-1α in STAT3-mediated trastuzumab resistance induction which would be valuable in designing more efficient chemosensitization strategies.

Yes1 signaling mediates the resistance to Trastuzumab/Lap atinib in breast cancer

BackgroundOverexpression of human epidermal growth factor receptor 2 (HER2) is observed in approximately 15–23% of breast cancers and these cancers are classified as HER2-positive breast cancer. Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer and has improved patient overall survival. However, acquired resistance to trastuzumab is still a critical issue in breast cancer treatment. We previously established a trastuzumab-resistant breast cancer cell line (named as BT-474-R) from a trastuzumab-sensitive HER2-amplified cell line BT-474. Lapatinib is also a molecular-targeted drug for HER2-positive breast cancer, which acquired the resistance to trastuzumab. Acquired resistance to lapatinib is also an issue to be conquered.MethodsWe established trastuzumab/lapatinib-dual resistant cell line (named as BT-474-RL2) by additionally treating BT-474-R with lapatinib. We analyzed the mechanisms of resistance to trastuzumab and lapatinib. Besides, we analyzed the effect of the detected resistance mechanism in HER2-positive breast cancer patients.ResultsProto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R and BT-474-RL2. Silencing of Yes1 by siRNA induced both BT-474-R and BT-474-RL2 to restore the sensitivity to trastuzumab and lapatinib. Pharmaceutical inhibition of Yes1 by the Src inhibitor dasatinib was also effective to restore the sensitivity to trastuzumab and lapatinib in the two resistant cell lines. Combination treatment with dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, the combination treatments induced G1-phase cell-cycle arrest and apoptosis. Consistent with cell line data, high expression of Yes1 mRNA was correlated with worse prognosis in patients with HER2-positive breast cancer.ConclusionYes1 plays an important role in acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancer. Our data suggest that pharmacological inhibition of Yes1 may be an effective strategy to overcome resistance to trastuzumab and lapatinib.

Neratinib resistance and cross-resistance to other HER2-targeted drugs due to increased activity of metabolism enzyme cytochrome P4503A4

Background:Neratinib is in Phase 3 clinical trials but, unfortunately, the development of resistance is inevitable. Here, we investigated the effects of acquired neratinib resistance on cellular phenotype and the potential mechanism of this resistance.Methods:Neratinib-resistant variants of HER2-positive breast cancer cells were developed and their cross-resistance investigated using cytotoxicity assays. Similarly, sensitivity of trastuzumab-resistant and lapatinib-resistant cells to neratinib was assessed. Cellular phenotype changes were evaluated using migration, invasion and anoikis assays. Immunoblotting for HER family members and drug efflux pumps, as well as enzyme activity assays were performed.Results:Neratinib resistance conferred cross-resistance to trastuzumab, lapatinib and afatinib. Furthermore, the efficacy of neratinib was reduced in trastuzumab- and lapatinib-resistant cells. Neratinib-resistant cells were more aggressive than their drug-sensitive counterparts, with increased CYP3A4 activity identified as a novel mechanism of neratinib resistance.Conclusions:The potential of increased CYP3A4 activity as a biomarker and/or target to add value to neratinib warrants investigation.

TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48. ©2016 AACR.

Superior Suppression of ErbB2-positive Tumor Cells by a Novel Human Triparatopic Tribody.

Downregulation of the epidermal growth factor receptor family of receptors is improved by combining different antibodies to noncompetitive epitopes. For ErbB2/HER2 this has already been translated into clinical practice by using a combination of trastuzumab and pertuzumab. Moreover, cocktails of 2 or 3 anti-epidermal growth factor receptor antibodies show an enhanced downregulation of the receptor due to the induction of matrix formation. A more efficient method for inducing matrix formation and receptor downregulation might include the use of trispecific reagents. A triparatopic Tribody consisting of 3 noncompeting ErbB2 binders was compared with equivalent trivalent monoparatopic counterparts, as well as to a cocktail of 3 monoclonal antibodies for its effects on downregulation of the ErbB2 receptor's kinase activity and survival of several ErbB2-expressing cancer cell lines. The triparatopic Tribody was significantly more efficient in downregulating ErbB2 and inhibiting tumor cell growth than either the control monoparatope tribodies or the combinatorial treatment with the 3 different parental antibodies on all the tested tumor cells, including trastuzumab-resistant cell lines. The enhancement of effectivity was dependent on all 3 binding moieties. Because the novel Tribody allows reduction of the costs of production (as only 1 construct provides the antitumor effects of 3 antibodies) and has an intermediate molecular size (∼100 kDa) well suited for both tumor penetration and acceptable half-life, it has the potential to become a precious tool for therapeutic use particularly in trastuzumab-resistant cancer patients.

MiR-129-5p Sensitizes the Response of Her-2 Positive Breast Cancer to Trastuzumab by Reducing Rps6

Background/Aims: Trastuzumab is an important treatment used for patients with Her-2-positive breast cancer, but an increasing incidence of trastuzumab resistance has been observed clinically during the past decade. Aberrant microRNA (miR) expression levels are correlated with prognosis and response to trastuzumab in breast cancer. MiR-129-5p is downregulated in trastuzumab-resistant human breast cancer cells (JIMT-1), but its potential function and underlying mechanism remain unclear. Methods: Quantitative RT-PCR (qRT-PCR) was used to determine the expression levels of miR-129-5p and its potential target genes. The effects of miR-129-5p on cell responses to trastuzumab were analyzed by CCK-8 and flow cytometry assays in Her-2-positive breast cancer cells (SKBR-3 and JIMT-1). Bio-informatics analyses were performed to predict target genes of miR-129-5p, and luciferase assays were carried out to confirm the binding of miR-129-5p and rpS6. Results: MiR-129-5p, which was downregulated and predicted to target rpS6 in trastuzumab-resistant breast cancer cells, enhanced the sensitivity of breast cancer cells to trastuzumab by reducing the expression of rpS6. Moreover, the overexpression of rpS6 reversed the sensitivity of cells to trastuzumab induced by miR-129-5p. Conclusions: MiR-129-5p sensitized Her-2-positive breast cancer to trastuzumab by downregulating rpS6. These findings provide novel insights into the common role of rpS6 and its related molecular mechanisms in mediating trastuzumab-resistance in Her-2-positive breast cancers.