Toru Mukohara

Gefitinib Induces Apoptosis in the EGFRL858R Non–Small-Cell Lung Cancer Cell Line H3255

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) have recently been described in patients with non–small-cell lung cancer (NSCLC) who achieve radiographic regressions to the EGFR inhibitor gefitinib. One of these mutations, L858R (Leu→Arg), is also found in NSCLC cell line H3255, which is very sensitive to gefitinib treatment. We characterized nine NSCLC cell lines (three isolated from patients with bronchioloalveolar carcinoma and six isolated from patients with adenocarcinoma) for their in vitro sensitivity to gefitinib. Of these, only H3255 (EGFRL858R) and H1666 (EGFRWT) are sensitive to gefitinib with IC50 values of 40 nmol/L and 2 μmol/L, respectively. We examined the effects of gefitinib on H3255 and cell lines containing wild-type EGFR that are either sensitive (H1666) or resistant (A549 and H441) to gefitinib exposure in vitro. Gefitinib treatment (1 μmol/L) leads to significant apoptosis accompanied by increased poly(ADP-ribose) polymerase cleavage only in the H3255 cell line, leads to G1-S arrest in H1666, and has no effects in the A549 and H441 cell lines. Although EGFR and AKT are constitutively phosphorylated in H3255, H1666, and H441 cell lines, AKT is completely inhibited by gefitinib treatment only in the H3255 cell line. These findings further characterize a mechanism by which gefitinib treatment of NSCLC harboring EGFRL858R leads to a dramatic response to gefitinib.

Association between gain-of-function mutations in PIK3CA and resistance to HER2-targeted agents in HER2-amplified breast cancer cell lines

BACKGROUND The mechanism of resistance to human epidermal growth factor receptor 2 (HER2)-targeted agents has not been fully understood. We investigated the influence of PIK3CA mutations on sensitivity to HER2-targeted agents in naturally derived breast cancer cells. MATERIALS AND METHODS We examined the effects of Calbiochem (CL)-387,785, HER2 tyrosine kinase inhibitor, and trastuzumab on cell growth and HER2 signaling in eight breast cancer cell lines showing HER2 amplification and trastuzumab-conditioned BT474 (BT474-TR). RESULTS Four cell lines with PIK3CA mutations (E545K and H1047R) were more resistant to trastuzumab than the remaining four without mutations (mean percentage of control with 10 microg/ml trastuzumab: 58% versus 92%; P = 0.010). While PIK3CA-mutant cells were more resistant to CL-387,785 than PIK3CA-wild-type cells (mean percentage of control with 1 microM CL-387,785: 21% versus 77%; P = 0.001), CL-387,785 retained activity against BT474-TR. Growth inhibition by trastuzumab and CL-387,785 was more closely correlated with changes in phosphorylation of S6K (correlation coefficient, 0.811) than those of HER2, Akt, or ERK1/2. Growth of most HER2-amplified cells was inhibited by LY294002, regardless of PIK3CA genotype. CONCLUSIONS PIK3CA mutations are associated with resistance to HER2-targeted agents. PI3K inhibitors are potentially effective in overcoming trastuzumab resistance caused by PIK3CA mutations. S6K phosphorylation is a possibly useful pharmacodynamic marker in HER2-targeted therapy.

Mechanisms of resistance to anti‐human epidermal growth factor receptor 2 agents in breast cancer

Approximately 20% of breast cancers are characterized by overexpression of human epidermal growth factor receptor 2 (HER2) protein and associated gene amplification, and the receptor tyrosine kinase is believed to play a critical role in the pathogenesis of these tumors. The development and implementation of trastuzumab, a humanized monoclonal antibody against the extracellular domain of HER2 protein, has significantly improved treatment outcomes in patients with HER2‐overexpressing breast cancer. However, despite this clinical usefulness, unmet needs for better prediction of trastuzumab’s response and overcoming primary and acquired resistance remain. In this review, we discuss several potential mechanisms of resistance to trastuzumab that have been closely studied over the last decade. Briefly, these mechanisms include: impaired access of trastuzumab to HER2 by expression of extracellular domain‐truncated HER2 (p95 HER2) or overexpression of MUC4; alternative signaling from insulin‐like growth factor‐1 receptor, other epidermal growth factor receptor family members, or MET; aberrant downstream signaling caused by loss of phosphatase and tensin homologs deleted from chromosome 10 (PTEN), PIK3CA mutation, or downregulation of p27; or FCGR3A polymorphisms. In addition, we discuss potential strategies for overcoming resistance to trastuzumab. Specifically, the epidermal growth factor receptor/HER2 tyrosine kinase inhibitor lapatinib partially overcame trastuzumab resistance in a clinical setting, so its efficacy results and limited data regarding potential mechanisms of resistance to the drug are also discussed. (Cancer Sci 2011; 102: 1–8)

Sensitivity of breast cancer cell lines to the novel insulin-like growth factor-1 receptor (IGF-1R) inhibitor NVP-AEW541 is dependent on the level of IRS-1 expression

To investigate the potential value of targeting insulin-like growth factor-1 receptor (IGF-1R) in breast cancer, we examined the effects of NVP-AEW541, a selective small-molecule inhibitor of the IGF-1R tyrosine kinase, in a panel of 16 breast cancer cell lines. All cell lines expressed IGF-1R, but MCF-7 expressed much higher levels of insulin receptor substrate-1 (IRS-1) than the others. NVP-AEW541 was more potent at inhibiting growth of MCF-7 cells as compared to the others (IC(50), 1 microM vs. approximately 7 microM). Comparing MCF-7 to T47D cells, which express IGF-1R at a level identical to MCF-7 but have less than 1/30 the amount of IRS-1, NVP-AEW541 caused cell-cycle arrest at the G1-S boundary, reduced in vitro cell migration, and enhanced the cytotoxic effects of vinorelbine and paclitaxel in MCF-7, but not in T47D. While NVP-AEW541 decreased the phosphorylation of IGF-1R in both cell lines, it inhibited phosphorylation of Akt and disrupted the IRS-1/PI3K complex only in MCF-7. These findings suggest that inhibiting IGF-1R may be an effective therapeutic strategy for breast cancers that co-express IGF-1R and IRS-1 at high levels.

Effect of axitinib (AG-013736) on fatigue, thyroid-stimulating hormone, and biomarkers: A phase I study in Japanese patients

Axitinib is an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor (VEGFR) 1, 2, and 3. This phase I study evaluated the safety, pharmacokinetics, pharmacodynamics, antitumor activity, and recommended starting dose of axitinib in patients with advanced solid tumors. Twelve patients received single‐dose axitinib 5 mg and were monitored for ≥48 h. Continuous 5 mg twice‐daily dosing was then initiated. One patient had dose‐limiting toxicity (grade 3 proteinuria and fatigue). Common treatment‐related adverse events were anorexia, fatigue, and diarrhea. Grade 3 treatment‐related adverse events were fatigue and hypertension. Maximum axitinib plasma concentration occurred 1–4 h after steady‐state dosing. Eleven patients experienced thyroid‐stimulating hormone elevation; time‐course change and fatigue onset appeared to be related in some patients. Significant correlation was observed between thyroid‐stimulating hormone change and area under the plasma concentration–time curve (AUC; r = 0.80, P = 0.005). Axitinib decreased plasma soluble vascular endothelial growth factor receptor 2 (s‐VEGFR2), with significant correlation between change in s‐VEGFR2 and AUC (r = −0.92, P < 0.0001). Fluorodeoxyglucose positron emission tomography revealed a substantial decrease in tumor metabolic activity associated with axitinib. Tumor size decreased in nine patients. The time‐course of thyroid‐stimulating hormone change appeared correlated with fatigue. There were significant correlations between thyroid‐stimulating hormone or s‐VEGFR2 and axitinib exposure. Axitinib 5 mg twice‐daily is the recommended starting dose for Japanese patients. This trial is registered with ClinicalTrials.gov, identifier NCT00447005. (Cancer Sci 2010)

PI3K mutations in breast cancer: prognostic and therapeutic implications

The PI3K pathway is the most frequently enhanced oncogenic pathway in breast cancer. Among mechanisms of PI3K enhancement, PIK3CA mutations are most frequently (∼30%) observed, along with protein loss of PTEN. Since the first discovery of PIK3CA mutations in solid malignancies in 2004, numerous studies have revealed the prognostic and therapeutic implications of these mutations. Although many issues remain unconfirmed, some have been carved in stone by the level of consistency they have shown among studies: 1) PIK3CA mutations are most likely to be observed in ER-positive/HER2-negative tumors, and are associated with other good prognostic characters; 2) PIK3CA mutations can coexist with other PI3K-enhancing mechanisms, such as HER2 amplification and PTEN protein loss; 3) PIK3CA mutations are potentially a good prognostic marker; 4) PIK3CA may predict a poorer tumor response to trastuzumab-based therapies, but its impact on disease-free survival and overall survival is uncertain; and 5) based on reports of early clinical trials, PIK3CA mutations do not guarantee a dramatic response to PI3K inhibitors. Collectively, there is currently no sufficient evidence to recommend routine genotyping of PIK3CA in clinical practice. Given that PIK3CA-mutant breast cancer appears to have a distinct tumor biology, development of more individualized targeted therapies based on the PIK3CA genotype is awaited.

Phase I and Pharmacokinetic Study of ABI-007, Albumin-bound Paclitaxel, Administered Every 3 Weeks in Japanese Patients with Solid Tumors

Objective ABI-007 is a novel Cremophor® EL-free nanoparticle albumin-bound paclitaxel. This Phase I study was designed to evaluate tolerability and determine recommended dose for Japanese patients when ABI-007 was administered in every-3-week schedule. Pharmacokinetics of paclitaxel was also assessed. Methods Patients with advanced solid tumors refractory to standard therapy received a 30 min intravenous infusion of ABI-007 every 3 weeks without pre-medications at 200, 260 or 300 mg/m2, respectively. Tolerability and recommended dose were determined by the standard ‘3 + 3’ rule. Results No dose-limiting toxicity was observed, despite the dose escalation. In another cohort, 260 mg/m2 was re-evaluated and resulted in no dose-limiting toxicity. Grade 3 or 4 neutropenia was reported for the majority of patients (n = 8) but no incidence of febrile neutropenia. Non-hematological toxicities were generally mild except for Grade 3 sensory neuropathy (n = 3). Pharmacokinetic study demonstrated the area under the curve of paclitaxel increased with increasing the dosage, and comparable pharmacokinetic parameters to the western population. Partial response was observed in three non-small cell lung cancer patients. Two of whom had received docetaxel-containing chemotherapy prior to the study. Conclusions ABI-007 administered in every-3-week schedule was well tolerated up to 300 mg/m2, and recommended dose was determined at 260 mg/m2 in consideration of efficacy, toxicities and similarity of pharmacokinetic profile in western studies. Additional studies of single-agent ABI-007 as well as platinum-based combinations, particularly in patients with non-small cell lung cancer, are warranted.

Therapeutic Targeting of Multiple Signaling Pathways in Malignant Pleural Mesothelioma

The majority of malignant pleural mesotheliomas (MPMs) aberrantly express the epidermal growth factor receptor (ErbB1). We examined the efficacy of GW572016 (lapatinib), a dual inhibitor of ErbB1/ErbB2 with a panel of 10 MPM cell lines. Two of the 10 MPM cell lines, H2373 and H2452, underwent G1/S cell cycle arrest and growth inhibition with an IC50 of 1 µM and 0.8 µM, respectively. There was no relationship between the presence or the amount of ErbB1, phospho-ErbB1, phospho-ErbB2, ErbB3, ErbB4, phospho-Akt, and Akt or the ability of lapatinib to inhibit phospho-ErbB1 in these cell lines compared to those that did not respond to lapatinib. The sensitive cell lines had a time-dependent decrease in phospho-Akt and/or ERK1/2, and an increase in p27 and when treated with lapatinib. The combination of lapatinib with U0126, LY294002 or rapamycin caused greater growth inhibition than either drug alone in the sensitive cell lines while this did not occur in the resistant cell lines. Our findings suggest that ErbB1 alone is a therapeutic target for the minority of mesotheliomas and that combining ErbB1 inhibitors with signal transduction inhibitors in mesothelioma will enhance their effectiveness. Furthermore, combinations of growth factor and signal transduction inhibitors may be needed to inhibit the growth of the majority of MPM cell lines, and therefore patients with MPM.

Absence of primary cilia in cell cycle-arrested human breast cancer cells

Previous studies using cultured cells showed that primary cilia are present in quiescent cells, but are absent in proliferating cells. We studied here the relationship between the presence or absence of primary cilia and the cell cycle arrest of normal epithelial cells and cancer cells in the human normal breast and breast cancer tissues. In normal breast tissues, although most epithelial cells were nonproliferating as estimated by the immunofluorescence staining of the proliferation marker Ki‐67, primary cilia were present only in 20–40% of the epithelial cells. In breast cancer tissues, primary cilia were not observed in any of the breast cancer cells. Furthermore, primary cilia were hardly observed in the nonproliferating cancer cells in the orthotopic and metastatic human breast cancer xenograft tumors in mice. These results indicate that the absence of primary cilia does not necessarily represent the proliferating phases of normal epithelial cells and cancer cells.

Inhibition of the mTOR/S6K signal is necessary to enhance fluorouracil-induced apoptosis in gastric cancer cells with HER2 amplification.

The purpose of this study was to explore the effect of trastuzumab in enhancing the activity of chemotherapeutic agents and the molecular basis of this effect. Two gastric cancer cell types with HER2 amplification, one sensitive (NCI‑N87) and one insensitive (MKN-7) to trastuzumab, were tested for the effects of trastuzumab on cell growth and cell signaling using MTS assay and western blotting, respectively. Interaction between trastuzumab and chemotherapeutic agents (fluorouracil, doxorubicin, cisplatin and paclitaxel) was evaluated by the combination index (CI). Fluorouracil-induced apoptosis was evaluated using western blot for poly (ADP-ribose) polymerase (PARP). Trastuzumab decreased phosphorylation of S6K, showed synergistic effect with fluorouracil or doxorubicin, and increased fluorouracil-induced apoptosis in NCI-N87 cells, but not in MKN-7 cells. While the mTOR inhibitor everolimus enhanced fluorouracil-induced apoptosis in both HER2-amplified cell lines, this was not the case in the gastric cancer cell lines without HER2 amplification. Consistently, while the EGFR/HER2 inhibitor Cl-387,785 inhibited cell growth of MKN-7, this growth inhibition did not accompany decrease in phosphorylation of S6K, and the compound did not enhance fluorouracil-induced apoptosis. In summary, inhibition of the mTOR/S6K signal may be a key molecular event in enhancing fluorouracil-induced apoptosis specifically in gastric cancer cells with HER2 amplification. mTOR inhibitors may therefore be attractive alternative drugs in gastric cancers with HER2 amplification regardless of their sensitivity to trastuzumab.