Evaluation of proliferation and apoptosis markers in circulating tumor cells of women with early breast cancer who are candidates for tumor dormancy.

INTRODUCTION: Clinical dormancy is frequently observed in breast cancer. In the present study, we aimed to characterize circulating tumor cells (CTCs) in dormancy candidates (DC) with early breast cancer in terms of proliferation and apoptosis. METHODS: Cytospins of peripheral blood mononuclear cells (PBMCs) were obtained from DC (n = 122) who were disease-free for at least 5 years and from metastatic patients (n = 40) who relapsed more than 5 years after surgery. Sequential samples from eight DC (n = 36) who maintained a prolonged disease-free status and from eight DC (n = 27) presenting late relapse during follow-up, were also analyzed. PBMCs were triple stained with a pancytokeratin, antibody along with anti-Ki67 and anti-M30 antibodies as proliferation and apoptosis markers, respectively. RESULTS: CTCs were identified in 40 (33%) of 122 DC and in 15 (37.5%) of 40 metastatic patients. In total, twenty-five (62.5%) DC had exclusively dormant (Ki67(-)/M30(-)), seven (17.5%) had proliferative Ki67(+)/M30(-), four (10%) had apoptotic Ki67(-)/M30(+) and four (10%) had both phenotypes of proliferative and apoptotic CTCs. In comparison, 53.4% of CTC-positive metastatic patients had exclusively dormant and 46.6% had proliferative CTCs; none had apoptotic CTCs (P = 0.039). Among all CTCs detected in DC patients, 82.4% were dormant, whereas in the nondormant population, 32.5% were proliferative and 67.5% apoptotic. The respective percentages in metastatic patients were 59.1%, 100% and 0% (P <0.0001). Moreover, apoptotic CTCs prevailed among nondormant CTCs detected in sequential samples from DC who remained in a prolonged disease-free status compared to those presenting late relapse during follow-up (70.6% versus 43.5% (P = 0.0002)). CONCLUSIONS: The apoptotic index of CTCs is increased during clinical dormancy, whereas the proliferation index is increased on relapse. In addition, apoptotic CTCs are more frequently encountered during follow-up in DC patients who remain disease-free compared to those with subsequent late relapse, suggesting that monitoring proliferation and apoptosis in CTCs during clinical dormancy merits further investigation as a tool for predicting late disease recurrence.

Hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients.

INTRODUCTION: The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs. METHODS: The expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGF2), HIF-1alpha and phosphorylated-focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and confocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1alpha or pFAK antibodies, respectively. RESULTS: pFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1alpha was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1alpha and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. Vascular endothelial growth mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1alpha and VEGF2. CONCLUSIONS: The expression of pFAK, HIF-1alpha, VEGF and VEGF2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.

Efficacy of Lapatinib in Therapy-Resistant HER2-Positive Circulating Tumor Cells in Metastatic Breast Cancer.

BACKGROUND: To evaluate the efficacy of lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, in therapy-resistant HER2-positive CTCs in metastatic breast cancer (MBC). PATIENTS AND METHODS: Patients with MBC and HER2-positive CTCs despite disease stabilization or response to prior therapy, received lapatinib 1500 mg daily in monthly cycles, till disease progression or CTC increase. CTC monitoring was performed by immunofluorescent microscopy using cytospins of peripheral blood mononuclear cells (PBMCs) double stained for HER2 or EGFR and cytokeratin. RESULTS: A total of 120 cycles were administered in 22 patients; median age was 62.5 years, 15 (68.2%) patients were post-menopausal and 20 (90.1%) had HER2-negative primary tumors. At the end of the second course, HER2-positive CTC counts decreased in 76.2% of patients; the median number of HER2-positive CTCs/patient also declined significantly (p = 0.013), however the decrease was significant only among patients presenting disease stabilization (p = 0.018) but not among those with disease progression during lapatinib treatment. No objective responses were observed. All CTC-positive patients harbored EGFR-positive CTCs on progression compared to 62.5% at baseline (p = 0.054). The ratio of EGFR-positive CTCs/total CTCs detected in all patients increased from 17.1% at baseline to 37.6% on progression, whereas the mean percentage of HER2-negative CTCs/patient increased from 2.4% to 30.6% (p = 0.03). CONCLUSIONS: The above results indicate that lapatinib is effective in decreasing HER2-positive CTCs in patients with MBC irrespectively of the HER2 status of the primary tumor and imply the feasibility of monitoring the molecular changes on CTCs during treatment with targeted agents. TRIAL REGISTRATION: Clinical trial.gov NCT00694252.

Apoptotic circulating tumor cells in early and metastatic breast cancer patients.

The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (polycaspase kit: 8.1% vs. 47.4% of the total CTC number; P = 0.0001; M30-antibody: 32.1% vs. 76.63%; P = 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics.

Caveolin-1 regulates EGFR signaling in MCF-7 breast cancer cells and enhances gefitinib-induced tumor cell inhibition.

Caveolin-1, an essential protein constituent of caveolae, is involved in cell signaling through its association with various signaling molecules. Epidermal growth factor receptor (EGFR) interacts directly with caveolin-1 and this interaction functionally regulates EGFR tyrosine kinase activity. In this report we investigated the role of caveolin-1 overexpression on EGFR signaling in MCF-7 breast cancer cell line. We demonstrate here that although total EGFR expression levels are similar, EGFR phosphorylation is diminished in MCF-7/CAV1 cells compared to parental MCF-7 cells. In addition, MCF-7/CAV1 cells exhibit higher total and activated Akt levels. Moreover, MCF-7/CAV1 cells stimulated with EGF display higher EGFR and Akt phosphorylation associated with enhanced proliferative and motility rates, compared to MCF-7 cells. Pretreatment with gefitinib inhibits EGF-induced stimulation of both EGFR and downstream Akt and MAPK more efficiently in MCF-7/CAV1 than in MCF-7 cells. In accordance, EGF-induced proliferation and migration is more effectively suppressed with gefinitib in MCF-7/CAV1 compared to parental cells. In conclusion these results suggest that caveolin-1 overexpression in MCF-7 breast cancer cell line modulates EGFR activation levels and EGF-induced EGFR signalling. This is associated with enhanced antitumor effects of gefitinib suggesting a role for EGFR inhibition in caveolin-1 overexpressing breast cancers.

Activation of FAK/PI3K/Rac1 signaling controls actin reorganization and inhibits cell motility in human cancer cells.

We have recently identified a specific signaling pathway that regulates actin reorganization in malignant human breast and prostate epithelial cells associated with FAK, PI-3K and Rac1 activation. Here we report that this pathway operates in MCF7 cells upon activation of membrane androgen receptors (mAR). Stimulation of mAR by the non-permeable testosterone-BSA conjugate resulted in early actin reorganization documented by quantitative measurements of actin dynamics and morphological analysis of microfilament organization. This effect was regulated by early phosphorylation of FAK and subsequent PI-3K and Rac1 activation. The functional role of this pathway was further shown in A375 melanoma cells. Treatment with the opioid antagonist alpha(s1) casomorphin resulted in rapid and potent actin remodeling in A375 cells, regulated by rapid activation of the FAK/PI-3K/Rac1 signaling. Pretreatment of both cell lines with the specific PI-3K inhibitor wortmannin blocked actin reorganization. Interestingly, wound healing assays revealed that testosterone-BSA and alpha (s1) casomorphin significantly inhibited MCF7 and A375 cell motility respectively. These effects were abrogated through blockade of PI-3K signaling by wortmannin. The results presented here indicate that actin reorganization through FAK/PI3-K/Rac-1 activation operates in various human cancer cell systems supporting a functional role for FAK/PI-3K/Rac1/actin signaling in controlling cell motility.