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Evidence is mounting that the occurrence of the CD44(pos)/CD24(neg/low) cell population, which contains potential breast cancer (BC) stem cells, could explain BC clinical resistance to HER2-targeted therapies. We investigated whether de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin) may relate to the dynamic regulation of the mesenchymal CD44(pos)/CD24(neg/low) phenotype in HER2-positive BC. We observed that the subpopulation of Tzb-refractory JIMT-1 BC cells exhibiting CD44(pos)/CD24(neg/low)-surface markers switched with time. Low-passage JIMT-1 cell cultures were found to spontaneously contain approximately 10% of cells bearing the CD44(pos)/CD24(neg/low) immunophenotype. Late-passage (>60) JIMT-1 cultures accumulated approximately 80% of CD44(pos)/CD24(neg/low) cells and closely resembled the CD44(pos)/CD24(neg/low)-enriched ( approximately 85%) cell population constitutively occurring in HER2-negative MDA-MB-231 mesenchymal BC cells. Dynamic expression of mesenchymal markers was not limited to CD44/CD24 because high-passages of JIMT-1 cells exhibited also reduced expression of the HER2 protein and over-secretion of pro-invasive/metastatic chemokines and metalloproteases. Accordingly, late-passage JIMT-1 cells displayed an exacerbated migratogenic phenotype in plastic, collagen, and fibronectin substrates. Intrinsic genetic plasticity to efficiently drive the emergence of the CD44(pos)/CD24(neg/low) mesenchymal phenotype may account for de novo resistance to HER2 targeting therapies in basal-like BC carrying HER2 gene amplification., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Clinicopathological assessment of the functional relationship between the HER2 oncogene and tumor-associated fatty acid synthase (FASN) is largely precluded because immunohistochemical and/or mRNA studies should be performed in biopsies from breast cancer patients. We here sought to determine whether serum FASN (sFASN) could associate with circulating HER2 extracellular domain (HER2 ECD) in the blood of metastatic breast cancer (MBC) patients. Concentrations of serum FASN and HER2 ECD were measured with ELISA in sera retrospectively obtained from 201 patients with metastatic breast cancer (MBC) and 31 healthy subjects. Mechanistical in vitro studies were performed using pharmacological inhibitors of HER2 and FASN as well as cultured cancer cells engineered to overexpress HER2 and FASN human genes. When the upper limit of normal sFASN was defined as the mean + 2SD of the control group, sFASN was elevated above this cut-off (12 ng/ml) in 70 MBC patients (35%). Eighty-nine MBC patients (44%) had elevated levels of HER2 ECD (HER2 ECD cut-off = 15 ng/ml). HER2 ECD-positive MBC patients slightly increased their sFASN levels compared with HER2 ECD-negative MBC patients. sFASN-positive MBC patients had significantly increased levels of HER2 ECD when compared with sFASN-negative MBC patients (mean HER2 ECD=34 ng/ml, 95% CI 26-41 ng/ml and 18 ng/ml -95% CI 15-21 ng/ml, respectively; p=0.002). Sixty percent of sFASN-positive patients concurrently exhibited high levels of HER2 ECD whereas 64% of sFASN-negative patients were negative for circulating HER2 ECD. In vitro studies revealed that BC cells bearing HER2 gene-amplification released higher levels of extracellular FASN than HER2-negative BC cells. Trastuzumab-induced blockade of HER2 ECD shedding failed to prevent FASN release and retrovirally-induced HER2 overexpression in MCF-7 cells did not increase extracellular FASN. Of note, pharmacological inhibition of FASN activity significantly decreased HER2 ECD levels in the supernatant of HER2-overexpressing BC cells while transient overexpression of FASN gene in HBL100 cells promoted FASN protein release and concomitantly increased HER2 ECD shedding into the extracellular milieu. Subsequent studies should explore if quantitative determination of FASN molecules in blood could become a rapid and accurate non-invasive test to monitor disease progression and survival in HER2-overexpressing MBC undergoing HER2-targeted therapies.

The small molecule HER2 tyrosine kinase inhibitor (TKI) lapatinib (Tykerb) is approved for the therapy of patients with HER2-positive breast carcinomas who have progressed on trastuzumab (Herceptin). Unfortunately, the efficacy of this HER2 TKI is limited by both primary (inherent) and acquired resistance, the latter typically occurring within 12 months of starting therapy. One of the key factors limiting our understanding of the mechanisms involved in lapatinib resistance is the lack of published preclinical models. We herein review lapatinib-refractory models recently developed at the bench and the survival pathways discovered. As hyperactivation of the pharmacologically targetable PI3K/mTOR/p70S6K1 axis appears to be central to the occurrence of lapatinib resistance, preclinical data showing enhanced antitumour effects when combining lapatinib with mTOR inhibitors (e.g., rapamycin analogues and NVP-BEZ235) highlight the importance of translational work to yield clinically useful regimens capable of delaying or treating lapatinib resistance. The unexpected ability of the anti-type II diabetes drug metformin to inactivate mTOR and decrease p70S6K1 activity further reveals that this biguanide, generally considered non-toxic and remarkably inexpensive, might be considered for new combinatorial lapatinib-based protocols in HER2-overexpressing breast cancer patients.

Introduction: This phase II study assessed the efficacy and safety of oral vinorelbine given weekly in combination with carboplatin (CBDCA) AUC 5 once every 3 weeks for four cycles in chemonaive patients with advanced non-small cell lung carcinoma (NSCLC), followed by consolidation therapy with single-agent oral vinorelbine in non-progressive patients., Methods: Chemonaive advanced NSCLC patients received four cycles of combination therapy with CBDCA AUC 5 day 1 and oral vinorelbine, 60 mg/m2 on days 1, 8 and 15 (cycle 1), dose increased to 80 mg/m2 (cycles 2-4) in absence of grades 3-4 neutropenia (NCI-CTCv2). Consolidation therapy with oral vinorelbine was continued (cycle 5) at same dosage; if dose was decreased during combination therapy, it was given at 60 mg/m2, then increased at 80 mg/m2 (cycle 6) in absence of grades 3-4 neutropenia until PD, toxicity or patient's refusal., Results: A total of 57 patients were registered and 56 patients were treated (ITT population), median age was 61 years (37-71). Objective response evaluated by RECIST was 17.9% (95% confidence interval, CI [8.9-30.4]) and disease control (CR, PR, NC) 73.2% (95% CI [59.7-84.2]), median progression-free survival 4.3 months (95% CI [3.1-5.1]) with median overall survival 9.7 months (95% CI [7.7-11.9]) and 1-year survival 37.1% (95% CI [24.4, 49.9]). Grades 3-4 neutropenia occurred in 67.9% of patients during combination and 20% during consolidation; febrile neutropenia occurred in 4 patients (7.1%) during combination therapy. Non-hematological toxicities occurred primarily during combination (grade 3 nausea and grade 3 vomiting in 7.1% of patients)., Conclusions: The combination of oral vinorelbine given weekly in 3-week cycles in combination with carboplatin followed by consolidation therapy with oral vinorelbine as a single-agent was able to achieve efficacy results in line with other doublets including carboplatin in terms of response as well as survival. This regimen reported a good profile of tolerability in the treatment of advanced NSCLC, allowing that this combination can be easily proposed for the palliative care of NSCLC patients where the advantages of carboplatin over cisplatin are still appreciated.

Three prominent hallmarks of triple-negative/basal-like breast carcinomas, a subtype of breast cancer gene phenotype associated with poor relapse-free and overall survival, are overexpression of the epidermal growth factor receptor (EGFR), hyperactivation of the MEK/ERK transduction pathway and high sensitivity to DNA-damaging agents. The cytotoxic interaction between EGFR inhibitors (monoclonal antibodies such as cetuximab and small molecule tyrosine kinase inhibitors such as gefitinib) and DNA cross-linking agents (e.g. platinum derivatives) might represent a promising combination for the treatment of triple-negative/basal-like breast tumors that are dependent upon EGFR/MEK/ERK signaling. We evaluated the growth and molecular interactions of the anti-EGFR antibody cetuximab (erbitux) and the DNA cross-linking agent cisplatin (cis-diammedichloroplatinum; CDDP) in the gefitinib-resistant MDA-MB-468 breast cancer cell line, an in vitro model system that shows many of the recurrent basal-like molecular abnormalities including ER-PR-HER2-negative status, TP53 deficiency, EGFR overexpression, PTEN loss and constitutive activation of the MEK/ERK pathway. Unlike other basal-like breast cancer models, MDA-MB-468 cells do not carry mutations of the key DNA repair gene BRCA1. Concurrent treatment with sub-optimal doses of cetuximab significantly enhanced CDDP-induced apoptotic cell death. However, an isobologram-based mathematical assessment of the nature of the interaction revealed a loss of synergism when employing a high-dose of cetuximab. Since BRCA1 depletion has been found to decrease DNA damage repair and cell survival in MDA-MB-468 cells when treated with DNA-damaging drugs, we employed ELISA-based quantitative analyses to measure BRCA1 protein levels in CDDP+/- cetuximab-treated cells. Cetuximab as single agent was as efficient as CDDP at increasing BRCA1 protein expression. Interestingly, cetuximab co-exposure significantly antagonized the ability of CDDP to up-regulate BRCA1 expression. Low-scale phosphor-proteomic approaches [i.e. phospho-receptor tyrosine kinase (RTK) and phospho-mitogen-activated protein kinases (MAPKs) Array Proteome Profiler capable of simultaneously identifying the relative levels of phosphorylation of 42 different RTKs and 23 different MAPKs and other serine/threonine kinases, respectively] revealed the ability of Cetuximab, as single agent, to paradoxically induce hyper-phosphorylation of EGFR while concomitantly deactivating p42/44 (ERK1/ERK2) MAPK. Unexpectedly, ELISA-based quantitative analyses of EGFR protein content demonstrated that simultaneous exposure to cetuximab and optimal doses of CDDP completely depleted EGFR protein in MDA-MB-468 cells. Although these findings preclinically support, at least in part, ongoing clinical trials for 'triple-negative/basal-like' metastatic breast cancer patients who are receiving either cetuximab alone versus cetuximab plus carboplatin (http://www.clinicaltrials.gov/ct/show/NCT00232505), the unexpected ability of CDDP to promote a complete depletion of the cetuximab target EGFR further suggests that treatment schedules, cetuximab/CDDP doses and BRCA1 status should be carefully considered when combining anti-EGFR antibodies and platinum derivatives in triple-negative/basal-like breast carcinomas.