The Novel Oral mTORC1/2 Inhibitor TAK-228 Reverses Trastuzumab Resistance in HER2-Positive Breast Cancer Models.

The use of anti-HER2 therapies has significantly improved clinical outcome in patients with HER2-positive breast cancer, yet a substantial proportion of patients acquire resistance after a period of treatment. The PI3K/AKT/mTOR pathway is a good target for drug development, due to its involvement in HER2-mediated signalling and in the emergence of resistance to anti-HER2 therapies, such as trastuzumab. This study evaluates the activity of three different PI3K/AKT/mTOR inhibitors, i.e., BEZ235, everolimus and TAK-228 in vitro, in a panel of HER2-positive breast cancer cell lines with primary and acquired resistance to trastuzumab. We assess the antiproliferative effect and PI3K/AKT/mTOR inhibitory capability of BEZ235, everolimus and TAK-228 alone, and in combination with trastuzumab. Dual blockade with trastuzumab and TAK-228 was superior in reversing the acquired resistance in all the cell lines. Subsequently, we analyse the effects of TAK-228 in combination with trastuzumab on the cell cycle and found a significant increase in G0/G1 arrest in most cell lines. Likewise, the combination of both drugs induced a significant increase in apoptosis. Collectively, these experiments support the combination of trastuzumab with PI3K/AKT/mTOR inhibitors as a potential strategy for inhibiting the proliferation of HER2-positive breast cancer cell lines that show resistance to trastuzumab.

The Hippo Pathway Transducers YAP1/TEAD Induce Acquired Resistance to Trastuzumab in HER2-Positive Breast Cancer.

Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance.

Autocrine CCL5 Effect Mediates Trastuzumab Resistance by ERK Pathway Activation in HER2-Positive Breast Cancer.

HER2-positive breast cancer is currently managed with chemotherapy in combination with specific anti-HER2 therapies, including trastuzumab. However, a high percentage of patients with HER2-positive tumors do not respond to trastuzumab (primary resistance) or either recur (acquired resistance), mostly due to molecular alterations in the tumor that are either unknown or undetermined in clinical practice. Those alterations may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor proliferation and metastasis. Using continued exposure of a HER2-positive cell line to trastuzumab, we generated a model of acquired resistance characterized by increased expression of several cytokines. Differential gene expression analysis indicated an overexpression of 15 genes, including five different chemokines, and highlighting CCL5/RANTES as the most overexpressed one. Functional studies, either by in vitro gene silencing or by in vitro and in vivo pharmacologic inhibition of the CCL5/CCR5 interaction with maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance to trastuzumab, which was mediated by ERK activation. In patient samples, increased CCL5 expression significantly correlated with lower rates of complete response after neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA. Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was statistically associated with worse disease-free survival and overall cancer survival in patients with early HER2-positive breast cancer.

Recent Insights into the Development of Preclinical Trastuzumab- Resistant HER2+ Breast Cancer Models.

BACKGROUND: Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy. OBJECTIVE: In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer. CONCLUSION: Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer.

Generation, characterization, and maintenance of trastuzumab-resistant HER2+ breast cancer cell lines.

Trastuzumab became the therapy of choice for patients with HER2-positive breast cancer in 1998, and it has provided clinical benefit ever since. However, a significant percentage of patients show primary resistance to trastuzumab at diagnosis, and most patients with metastatic disease that initially respond to trastuzumab eventually progress (acquired resistance). Consequently, there is an urgent need to improve our knowledge of the mechanisms governing resistance, so that specific therapeutic strategies can be developed to provide improved efficacy. We generated new cell lines derived from BCCL through extended exposure to trastuzumab. Drug-conditioned populations were authenticated for their molecular profile and their resistance rate was determined. Heterogeneous HER2 amplification was observed across most of the BCCLs, ranging from cells without HER2 amplification to elevated HER2 gene copy numbers in others. Using a phospho-antibody array we analyzed the status of kinase receptors and effectors from different cellular pathways. This revealed that HER2, AKT, and S6RP presented high phosphorylation levels with specific variations between sensitive and resistant populations. In addition, differences in phosphorylation levels for several of those pathways targets were found between sensitive and resistant lines. Furthermore, a biochemical study characterized patterns of molecular alterations similar to those commonly described in breast cancer. Finally, a subcutaneous xenograft murine model confirmed the resistance to trastuzumab of the established cell line. We conclude that these resistant BCCLs can be a valuable tool to gain insight into the mechanisms of acquisition of trastuzumab resistance.

c-Jun N-Terminal Kinase Inactivation by Mitogen-Activated Protein Kinase Phosphatase 1 Determines Resistance to Taxanes and Anthracyclines in Breast Cancer.

MAPK phosphatase-1 (MKP-1) is overexpressed during malignant transformation of the breast in many patients, and it is usually associated with chemoresistance through interference with JNK-driven apoptotic pathways. Although the molecular settings of the mechanism have been documented, details about the contribution of MKP-1 to the failure of chemotherapeutic interventions are unclear. Transient overexpression of MKP-1 and treatment with JNK-modulating agents in breast carcinoma cells confirmed the mediation of MKP-1 in the resistance to taxanes and anthracyclines in breast cancer, through the inactivation of JNK1/2. We next assessed MKP-1 expression and JNK1/2 phosphorylation status in a large cohort of samples from 350 early breast cancer patients treated with adjuvant anthracycline-based chemotherapy. We detected that MKP-1 overexpression is a recurrent event predominantly linked to dephosphorylation of JNK1/2 with an adverse impact on relapse of the tumor and overall and disease-free survival. Moreover, MKP-1 and p-JNK1/2 determinations in 64 locally advanced breast cancer patients treated with neoadjuvant taxane-based chemotherapy showed an inverse correlation between MKP-1 overexpression (together with JNK1/2 inhibition) and the pathologic response of the tumors. Our results emphasize the importance of MKP-1 as a potential predictive biomarker for a subset of breast cancer patients with worse outcome and less susceptibility to treatment. Mol Cancer Ther; 15(11); 2780-90. ©2016 AACR.

Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma.

Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

Antidepressant-like effect of the novel thiadiazolidinone NP031115 in mice.

Glycogen synthase kinase-3beta (GSK-3beta) is an enzyme that phosphorylates glycogen synthase, thereby inhibiting glycogen synthesis. Besides this role, it is now believed that this enzyme plays an important role in the pathophysiology of many brain diseases including depression. Some inhibitors of this enzyme have shown antidepressant effects in animal models. This study investigated the effects of a novel thiadiazolidinone NP031115, a putative GSK-3beta inhibitor, and the well-established GSK-3beta inhibitor AR-A014418 in the mouse forced swimming test (FST), a model widely used to evaluate antidepressant activity. We found that NP031115 had an IC50 of 1.23 and 6.5 microM for GSK-3beta and GSK-3alpha, respectively. NP031115 (0.5 and 5 mg/kg, i.p.), in a way similar to imipramine (15 mg/kg, i.p), fluoxetine (32 mg/kg, i.p), AR-A014418 (9 mg/kg, i.p.), and rosiglitazone (5 microg/site, i.c.v.), significantly reduced immobility time in the FST. NP031115 at the higher dose and AR-A014418 (9 mg/kg, i.p.) reduced locomotion in the open-field test. Rosiglitazone (30 microM), AR-A014418 (1 microM), PG(J2) (10 microM), and NP031115 (1, 10 and 25 microM) activate PPARgamma in CHO transfected cells. GW-9662 (10 microg/site, i.c.v, a PPARgamma antagonist) administered 15 min before NP03115 (5 mg/kg, i.p.) or co-administered with rosiglitazone (5 microg/site, i.c.v.) prevented the antidepressant-like effect of these drugs in the FST. The results of this study show that NP031115 can exhibit an antidepressant effect, likely by inhibiting GSK-3beta and enhancing PPARgamma activity.

Glycogen synthase kinase-3 (GSK-3) inhibitory activity and structure-activity relationship (SAR) studies of the manzamine alkaloids. Potential for Alzheimer's disease.

Manzamine A and related derivatives isolated from a common Indonesian sponge, Acanthostrongylophora, have been identified as a new class of GSK-3beta inhibitors. The semisynthesis of new analogues and the first structure-activity relationship studies with GSK-3beta are also reported. Moreover, manzamine A proved to be effective in decreasing tau hyperphosphorylation in human neuroblastoma cell lines, a demonstration of its ability to enter cells and interfere with tau pathology. Inhibition studies of manzamine A against a selected panel of five different kinases related to GSK-3beta, specifically CDK-1, PKA, CDK-5, MAPK, and GSK-3alpha, show the specific inhibition of manzamine A on GSK-3beta and CDK-5, the two kinases involved in tau pathological hyperphosphorylation. These results suggest that manzamine A constitutes a promising scaffold from which more potent and selective GSK-3 inhibitors could be designed as potential therapeutic agents for Alzheimer's disease.

Enhanced induction of the immunoproteasome by interferon gamma in neurons expressing mutant Huntingtin.

Huntington disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cell, suggested that alterations in the ubiquitin-proteasome system (UPS) might contribute to HD pathogenesis. In previous work we reported that in a conditional mouse model of Huntington's disease (HD94 mice), the chymiotrypsin- and trypsin-"like" activities of the proteasome are increased selectivity in the affected and aggregate-containing brain regions: striatum, and cortex. Moreover, in these areas a neuronal increase in the interferon-inducible subunits of the immunoproteasome LMP2 and LPM7 was observed. In order to test if the expression of N-terminal mutant huntingtin (htt) by itself is sufficient to induce the change in proteasome catalytic activities as well as in LMP2 subunit expression, we performed activities of the proteasome and western blot experiments in striatal cultured neurons from HD94 mice free of glial contamination. We found no changes in any of the activities in these cells. Furthermore, western blot analysis performed with specific antibody against LMP2 subunits, revealed no difference in levels of this subunit in striatal neurons from HD94 compared to control cultures were treated with interferon-gamma (IFN-gamma) during 72 hours, a clear increase in LMP2 levels was observed in control neuronal cultures. Interestingly, this increase was much more pronounced (95% higher) in HD94 striatal cultures. These results indicate that although expression of mutant htt is not sufficient to induce the changes in proteasome catalytic core observed in HD, it synergizes the changes induced by IFN-gamma. Furthermore, immunocytochemical studies revealed that HD94 striatal neuron expressing high levels of LMP2 subunit showed a pre-apoptotic appearance. These results suggest that the correlation between neuronal induction of the immunoproteasome and neurodegeneration found in HD brains is secondary to inflammatory processes.

Neuronal induction of the immunoproteasome in Huntington's disease.

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration.

Nuclear localization of N-terminal mutant huntingtin is cell cycle dependent.

Unlike normal huntingtin (htt) which is located predominantly in the cytoplasm, mutant htt is also found in the nucleus of affected neurons. Nuclear localization of toxic polyglutamine-containing proteins has been postulated to be necessary for the pathogenesis of triplet repeat disorders. However, little is known about the mechanism by which mutant htt enters the nucleus. We have recently reported exclusive nuclear localization of exon 1 mutant htt in striatal primary neuronal cultures from the HD94 conditional mouse model of HD. This seemed to contradict the predominant cytoplasmic localization of N-terminal htt reported from transfection experiments and prompted us to hypothesize that subcellular localization of the toxic htt fragment might be favoured in nondividing cells. To test this, we analyzed subcellular localization of mutant htt in HD94 mixed neuron-glia cultures. Subconfluent glial cells showed cytoplasmic localization. However, nuclear localization was prompted by confluence, by serum withdrawal, and by treatment with cell cycle progression inhibitors such as Ara C or lactacystin. BrdU labelling experiments further confirmed that nuclear localization does not occur in dividing cells. Our findings offer an explanation for the neuronal specific toxicity of mutant htt despite its ubiquitous expression. Unraveling the mechanism of this cell cycle arrest-dependent entrance into the nucleus may offer new opportunities for therapeutic intervention.

Ephrin-B1 promotes dendrite outgrowth on cerebellar granule neurons.

Ephrins are developmentally regulated molecules that may contribute to axonal pathfinding through their binding to Eph receptor tyrosine kinases. In many cases, ephrins act as negative molecules that stimulate growth cone collapse, although some forms may promote axonal growth. Here, we have addressed the role played by ephrin-B1 during rat postnatal cerebellar development. Ephrin-B1 is expressed by both granule and Purkinje neurons whereas EphB is present in granule neurons in early postnatal cerebellum at a time coincident with axonal and dendrite outgrowth. Stably transfected 3T3 cells overexpressing ephrin-B1 enhance survival and neurite growth from cultured cerebellar granule neurons, an effect that is inhibited by the presence of an excess of a soluble EphB protein. Ephrin-B1-induced neuritogenesis is correlated with an increased expression of certain neuronal-specific microtubule-associated proteins (MAPs). Cerebellar granule neurons plated on stably transfected 3T3 cells overexpressing ephrin-B1 show an up-regulation of the expression of axonal MAPs such as Tau and phosphorylated MAP2C compared with neurons cultured on control 3T3 cells. The level of expression of these axonal MAPs is similar to that found in neurons plated on poly-L-lysine. Interestingly, there is a noteworthy up-regulation of somatodendritic MAPs such as high-molecular-weight MAP2 and mode II-phosphorylated MAP1B in neurons cultured on stably transfected 3T3 cells overexpressing ephrin-B1 compared with neurons plated on either control 3T3 cells or poly-L-lysine. In view of these data, we suggest that ephrin-B1 favors dendritogenesis of granule neurons during cerebellum development.