Modulation of MicroRNA-194 and cell migration by HER2-targeting trastuzumab in breast cancer

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 3'-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.     

Up-regulation of miR-21 mediates resistance to trastuzumab therapy for breast cancer

Trastuzumab resistance emerges to be a major issue in anti-human epidermal growth factor receptor 2 (HER2) therapy for breast cancers. Here, we demonstrated that miR-21 expression was up-regulated and its function was elevated in HER2(+) BT474, SKBR3, and MDA-MB-453 breast cancer cells that are induced to acquire trastuzumab resistance by long-term exposure to the antibody, whereas protein expression of the PTEN gene, a miR-21 target, was reduced. Blocking the action of miR-21 with antisense oligonucleotides re-sensitized the resistant cells to the therapeutic activities of trastuzumab by inducing growth arrest, proliferation inhibition, and G(1)-S cell cycle checking in the presence of the antibody. Ectopic expression of miR-21 in HER2(+) breast cancer cells confers resistance to trastuzumab. Rescuing PTEN expression with a p3XFLAG-PTEN-mut construct with deleted miR-21 targeting sequence at its 3' UTR restored the growth inhibition of trastuzumab in the resistant cells by inducing PTEN activation and AKT inhibition. In vivo, administering miR-21 antisense oligonucleotides restored trastuzumab sensitivity in the resistant breast cancer xenografts by inducing PTEN expression, whereas injection of miR-21 mimics conferred trastuzumab resistant in the sensitive breast tumors via PTEN silence. Up-regulatin of miR-21 in tumor biopsies obtained from patients receiving pre-operative trastuzumab therapy was associated with poor trastuzumab response. Therefore, miR-21 overexpression contributes to trastuzumab resistance in HER2(+) breast cancers and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to anti-HER2 treatment.     

Houttuyninum, an active constituent of Chinese herbal medicine, inhibits phosphorylation of HER2/neu receptor tyrosine kinase and the tumor growth of HER2/neu-overexpressing cancer cells

AimsThe overexpression of HER2/neu receptor plays a key role in tumorigenesis and tumor progression. Small molecules targeting HER2/neu have therapeutic value in cancers that overexpress HER2. In this present study, the effect of houttuyninum, a component in the Chinese herbal medicine Houttuynia cordata Thunb, on HER2/neu tyrosine phosphorylation and its in vivo antitumour activity was investigated.Main methodsThe phosphorylation and expression of proteins were determined by Western blot analysis. The MTT assay was employed to examine the inhibition of cell proliferation in vitro. Xenografts were established in nude mice for evaluating the antitumour activity of houttuyninum in vivo.Key findingsHouttuyninum inhibited phosphorylation of HER2 in a dose-dependent manner with an IC50 of 5.52 μg/ml without reducing HER2/neu protein expression in MDA-MB-453 cells. Houttuyninum also inhibited the activation of ERK1/2 and AKT, downstream molecules in the HER2/neu-mediated signal transduction pathway. In contrast, tyrosine phosphorylation of EGFR was unaffected when the concentration of houttuyninum was increased to 40 μg/ml in both A431 cells and MDA-MB-468 cells. Additionally, houttuyninum preferentially inhibited the growth of MDA-MB-453 cells that overexpressed HER2/neu; the MDA-MB-468 cells that overexpress EGFR remained unaffected. Administration of houttuyninum in vivo resulted in a significant reduction of phosphorylated HER2 levels and in tumor volumes of the BT474 and N87 xenografts, which both overexpress HER2/neu.SignificanceOur findings showed that houttuyninum can inhibit the HER2/neu signalling pathway and the tumor growth of cancer cells that overexpress HER2/neu. This drug may provide therapeutic value in the treatment of cancers that involve overexpression of HER2/neu.     

Targeting of HER3 with Functional Cooperative miRNAs Enhances Therapeutic Activity in HER2-Overexpressing Breast Cancer Cells

Background

The HER3 receptor functions as a major cause of drug resistance in cancer treatment. It is believed that therapeutic targeting of HER3 is required to improve patient outcomes. It is not clear whether a novel strategy with two functional cooperative miRNAs would effectively inhibit erbB3 expression and potentiate the anti-proliferative/anti-survival effects of a HER2-targeted therapy (trastuzumab) and chemotherapy (paclitaxel) on HER2-overexpressing breast cancer cells.

Results

Combination of miR-125a and miR-205, as compared to either miRNA alone, potently inhibited expression of HER3 in HER2-overexpressing breast cancer BT474 cells. Co-expression of the two miRNAs not only reduced the levels of phosphorylated erbB3 (P-erbB3), Akt (P-Akt), and Src (P-Src), it also inhibited cell proliferation and increased cells at G1 phase. A multi-miRNA lentiviral vector - the cluster of miR-125a and miR-205 - was constructed to simultaneously express the two miRNAs in HER2-overexpressing breast cancer cells. Concurrent expression of miR-125a and miR-205 via the miRNA cluster transfection significantly enhanced trastuzumab-mediated growth inhibition and cell cycle G1 arrest in BT474 cells and markedly increased paclitaxel-induced apoptosis in another HER2-overexpressing breast cancer cell line HCC1954.

Conclusions

Here, we showed that functional cooperative miRNAs effectively suppressed erbB3 expression. This novel approach targeting of HER3 was able to enhance the therapeutic efficacy of trastuzumab and paclitaxel against HER2-overexpressing breast cancer.

     

Dysregulation of HER2/HER3 signaling axis in Epstein-Barr virus-infected breast carcinoma cells

The role of Epstein-Barr virus (EBV) in the pathogenesis of breast cancer has been of long-standing interest to the field. Breast epithelial cells can be infected by EBV through direct contact with EBV-bearing lymphoblastoid cells, and EBV infection has recently been shown to confer breast cancer cells an increased resistance to chemotherapeutic drugs. In this study, we established EBV-infected breast cancer MCF7 and BT474 cells and demonstrated that EBV infection promotes tumorigenic activity of breast cancer cells. Firstly, we showed that the EBV-infected MCF7-A and BT474-A cells exhibited increased anchorage-independent growth in soft agar. The increased colony formation capacity in soft agar was associated with increased expression and activation of HER2/HER3 signaling cascades, as evidenced by the findings that the treatment of HER2 antibody trastuzumab (Herceptin), phosphatidylinositol 3-kinase inhibitor, or MEK inhibitor completely abolished the tumorigenic capacity. In the EBV-infected breast cancer cells, the expression of EBV latency genes including EBNA1, EBER1, and BARF0 was detected. We next showed that BARF0 alone was sufficient to efficiently up-regulate HER2/HER3 expression and promoted tumorigenic activity in MCF7 and BT474 cells by the use of both overexpression and small interfering RNA knock-down. Collectively, we demonstrated that EBV-encoded BARF0 promotes the tumorigenic activity of breast cancer cells through activation of HER2/HER3 signaling cascades.     

Evaluating treatment response using DW-MRI and DCE-MRI in trastuzumab responsive and resistant HER2-overexpressing human breast cancer xenografts

We report longitudinal diffusion-weighted magnetic resonance imaging (DW-MRI) and dynamic contrast enhanced (DCE)-MRI (7 T) studies designed to identify functional changes, prior to volume changes, in trastuzumab-sensitive and resistant HER2 + breast cancer xenografts. Athymic mice (N = 33) were subcutaneously implanted with trastuzumab-sensitive (BT474) or trastuzumab-resistant (HR6) breast cancer cells. Tumor-bearing animals were distributed into four groups: BT474 treated and control, HR6 treated and control. DW- and DCE-MRI were conducted at baseline, day 1, and day 4; trastuzumab (10 mg/kg) or saline was administered at baseline and day 3. Animals were sacrificed on day 4 and tumors resected for histology. Voxel-based DW- and DCE-MRI analyses were performed to generate parametric maps of ADC, K^trans, and v_e. On day 1, no differences in tumor size were observed between any of the groups. On day 4, significant differences in tumor size were observed between treated vs. control BT474, treated BT474 vs. treated HR6, and treated vs. control HR6 (P < .0001). On day 1, v_e was significantly higher in the BT474 treated group compared to BT474 control (P = .002) and HR6 treated (P = .004). On day 4, v_e and K^trans were significantly higher in the treated BT474 tumors compared to BT474 controls (P = .0007, P = .02, respectively). A significant decrease in Ki67 staining reinforced response in the BT474 treated group compared to BT474 controls (P = .02). This work demonstrated that quantitative MRI biomarkers have the sensitivity to differentiate treatment response in HER2 + tumors prior to changes in tumor size.     

mRNA Profiling Reveals Determinants of Trastuzumab Efficiency in HER2-Positive Breast Cancer

Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs) affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we called and analyzed SNPs. These results contribute to a better understanding of trastuzumab action and resistance mechanisms.     

HER2 signaling downregulation by trastuzumab and suppression of the PI3K/Akt pathway: an unexpected effect on TRAIL-induced apoptosis

We investigated whether HER2 downregulation by trastuzumab modulates the responsiveness of breast cancer cells to TNF-related apoptosis-inducing ligand (TRAIL). Interestingly, in contrast to increased response to TRAIL in SKBr3 cells, trastuzumab decreased the susceptibility of BT474 cells to TRAIL. This decrease was also observed after exogenous inhibition of PI3-K/Akt kinase, but not MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK). In BT474 cells, but not SKBr3 cells, inhibition of the HER2/phosphatidylinositol 3' kinase (PI3K)/Akt pathway resulted in downregulation of the pro-apoptotic receptors TRAIL-receptor 1 (TRAIL-R1) and TRAIL-R2. TRAIL-induced caspase-8 activation, Bid processing, drop of DeltaPsi(m), and poly ADP-ribose polymerase (PARP) cleavage but not in caspase-9 activation, and these events were inhibited in HER2/PI3K/Akt-suppressed BT474 cells, which on the other hand exhibited downregulation of Bcl-xL and increased response to mitomycin C. We show that HER2/PI3K/Akt pathway may play a specific pro-apoptotic role in certain cell type by inducing TRAIL-R1 and -R2 expression and thereby enhancing responsiveness to TRAIL.     

A novel mouse monoclonal antibody targeting ErbB2 suppresses breast cancer growth

Overexpression of ErbB2 in breast cancer is associated with increased recurrence and worse prognosis. Accumulating evidences suggest that molecular targeted therapy is a promising anticancer strategy. In this study, we produced a novel anti-ErbB2 monoclonal antibody, 6G10, that recognized an epitope distinct from the trastuzumab binding site. 6G10 induced aggregation of BT474 breast cancer cells and inhibited proliferation of various breast cancer cell lines including BT474. A growth inhibition assay showed that 6G10 had EC₅₀ values comparable to trastuzumab, indicating that the drugs have a similar level of potency. Furthermore, intraperitoneal administration of 6G10 completely inhibited the growth of xenografted tumors derived from BT474 and SK-BR-3 cells. These data suggested that 6G10 has great therapeutic potential and could be administered to patients alternatively, or synergistically, with trastuzumab.     

89Zr-mAb3481 PET for HER3 tumor status assessment during lapatinib treatment

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 (⁸⁹Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on⁸⁹Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to ⁸⁹Zr-mAb3481 administration. Subsequently, ex vivo ⁸⁹Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). ⁸⁹Zr-mAb3481 BT474 tumor uptake was remarkably high at SUV_mean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg ⁸⁹Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect ⁸⁹Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. ⁸⁹Zr-mAb3481 PET accurately reflected HER3 tumor status. ⁸⁹Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.     

Target therapy in HER2-overexpressing breast cancer patients

The development of new therapeutic strategies, such as monoclonal antibodies directed against human epidermal growth factor receptor-2 (HER2), has offered new hopes for women with early breast cancer whose tumors overexpress HER2. We retrospectively analyzed the population-based data of Breast Cancer Registry of Palermo in 2004-2006, and selected 1401 invasive breast cancer cases, nonmetastatic at diagnosis, having HER2/neu oncogene expression determined. We have correlated this information to age, tumor stage at diagnosis (TNM), nodal involvement, and receptor status (ER and PgR). Survival analysis was conducted dividing the patients in two different groups according to date of diagnosis: one group diagnosed in 2004 and a second group in 2005-2006. In the 460 cases of 2004, nodal involvement, receptor status, age at diagnosis and TNM maintained a strong predictive value (p?相似文献   

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.     

Antitumor activity of Herceptin in combination with STEALTH liposomal cisplatin or nonliposomal cisplatin in a HER2 positive human breast cancer model

Single agent antitumor activity of Herceptin, a humanized monoclonal antibody directed against HER2, has been demonstrated in numerous preclinical and clinical studies. Additionally, combination therapy with Herceptin and chemotherapy (CRx) has demonstrated additive antitumor activity in both preclinical models and early clinical trials. STEALTH (pegylated) liposomal (PL) cisplatin, also known as SPI-077, is currently in clinical trials for a variety of solid tumors. The three studies reported here discuss the antitumor activity of the combination of Herceptin and nonliposomal cisplatin or PL-cisplatin in two xenograft tumor models, initiated from the cell lines, BT474 and MDA453, that overexpress the oncogene, HER2. Herceptin alone had significant antitumor activity in all three experiments (p < 0.0001). Nonliposomal cisplatin and PL-cisplatin were both effective antitumor agents but, at tolerable dose levels, PL-cisplatin was superior to nonliposomal cisplatin (p < 0.0003). The effect of combining Herceptin with the chemotherapeutic cisplatin or PL-cisplatin, was most significant at moderate doses of H (0.5 mg/kg, p < 0.0001), but tended to be greater than either agent alone in all experiments. The combination of PL-cisplatin with Herceptin had statistically similar antitumor activity to that of nonliposomal cisplatin with Herceptin in all experiments. We conclude that combination therapy with PL-cisplatin and Herceptin results in significant antitumor activity with the potential for reducing toxicity in metastatic breast cancer patients.     

Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

BACKGROUND: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. METHODS: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. RESULTS: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. CONCLUSION: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.     

Dual Inhibitor AEE788 Reduces Tumor Growth in Preclinical Models of Medulloblastoma

Medulloblastoma is the most frequent malignant pediatric brain tumor with a dismal prognosis in 30% of cases. We examined the activity of AEE788, a dual inhibitor of human epidermal receptor (HER) 1/2 and vascular endothelial growth factor receptor (VEGFR) 1/2, in medulloblastoma preclinical models. Established lines (Daoy and D283), chemoresistant (Daoy(Pt)), and ectopically HER2-overexpressing (Daoy(HER2)) cells expressed diverse levels of total and activated AEE788 target receptors. In vitro, AEE788 inhibited cell proliferation (IC(50) from 1.7 to 3.8 μM) and prevented epidermal growth factor- and neuregulin-induced HER1, HER2, and HER3 activation. Inhibition of Akt paralleled that of HER receptors. In vivo, AEE788 growth inhibited Daoy, Daoy(Pt), and Daoy(HER2) xenografts by 51%, 45%, and 72%, respectively. Immunohistochemical analysis of mock- and HER2-transfected xenografts revealed that the latter showed, along with high HER2 expression, high VEGFR2 staining in tumor and endothelial cells and increased expression of the endothelial marker CD31. AEE788 reduced the activation of target receptors and angiogenesis. In 21 primary medulloblastoma, HER2 expression significantly correlated (P < .01) with VEGFR2 (r = 0.56) and VEGF (r = 0.61). In conclusion, AEE788 shows similar growth-suppressive activities in chemosensitive and chemoresistant medulloblastoma cells in vitro and in vivo. Ectopic HER2 overexpression sensitizes cells to AEE788 in vivo, but not in vitro, possibly through host-mediated processes. Together with the experimental data, the finding that HER2 positively correlates with VEGFR2 and VEGF in human medulloblastoma specimens indicates HER2-overexpressing medulloblastoma as the subset that most likely might benefit from AEE788 treatment.     

Lapatinib Plasma and Tumor Concentrations and Effects on HER Receptor Phosphorylation in Tumor

Purpose

The paradigm shift in cancer treatment from cytotoxic drugs to tumor targeted therapies poses new challenges, including optimization of dose and schedule based on a biologically effective dose, rather than the historical maximum tolerated dose. Optimal dosing is currently determined using concentrations of tyrosine kinase inhibitors in plasma as a surrogate for tumor concentrations. To examine this plasma-tumor relationship, we explored the association between lapatinib levels in tumor and plasma in mice and humans, and those effects on phosphorylation of human epidermal growth factor receptors (HER) in human tumors.

Experimental Design

Mice bearing BT474 HER2+ human breast cancer xenografts were dosed once or twice daily (BID) with lapatinib. Drug concentrations were measured in blood, tumor, liver, and kidney. In a randomized phase I clinical trial, 28 treatment-naïve female patients with early stage HER2+ breast cancer received lapatinib 1000 or 1500 mg once daily (QD) or 500 mg BID before evaluating steady-state lapatinib levels in plasma and tumor.

Results

In mice, lapatinib levels were 4-fold higher in tumor than blood with a 4-fold longer half-life. Tumor concentrations exceeded the in vitro IC₉₀ (~ 900 nM or 500 ng/mL) for inhibition of HER2 phosphorylation throughout the 12-hour dosing interval. In patients, tumor levels were 6- and 10-fold higher with QD and BID dosing, respectively, compared to plasma trough levels. The relationship between tumor and plasma concentration was complex, indicating multiple determinants. HER receptor phosphorylation varied depending upon lapatinib tumor concentrations, suggestive of changes in the repertoire of HER homo- and heterodimers.

Conclusion

Plasma lapatinib concentrations underestimated tumor drug levels, suggesting that optimal dosing should be focused on the site of action to avoid to inappropriate dose escalation. Larger clinical trials are required to determine optimal dose and schedule to achieve tumor concentrations that maximally inhibit HER receptors.

Trial Registration

Clinical Trial Registration: NCT00359190     

Solamargine induces apoptosis and enhances susceptibility to trastuzumab and epirubicin in breast cancer cells with low or high expression levels of HER2/neu

Trastuzumab is used for breast cancer patients with high expression levels of HER2 (human epidermal growth factor receptor 2)/neu; however, it has no effect on cancers with low levels of HER2/neu. SM (solamargine), a major steroidal alkaloid glycoside purified from Solanum incanum, triggered apoptosis of breast cancer cells (MCF-7 and SK-BR-3 cells) and non-cancerous breast epithelial cells (HBL-100 cells) within 3 h. To extend the application of trastuzumab in breast cancer patients, the regulation of HER2/neu expression by SM was investigated. SM significantly up-regulates HER2/neu expression in breast cancer cells with low and high expression levels of HER2/neu, and synergistically enhanced the effect of trastuzumab in inhibiting cell proliferation. Additionally, HER2/neu and TOP2A [TopoII (topoisomerase II) alpha] genes share the same amplicon on an identical chromosome. Notably, SM co-regulates HER2/neu and TopoIIalpha expression markedly, and enhances TopoII inhibitor-EPI (epirubicin)-induced cytotoxicity to breast cancer cells.     

Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.     

S100A14, a Member of the EF-hand Calcium-binding Proteins,Is Overexpressed in Breast Cancer and Acts as a Modulator of HER2 Signaling

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK. S100A14, one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens, and there is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding. Moreover, we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.