Daniele Generali

Up-regulation of Delta-like 4 Ligand in Human Tumor Vasculature and the Role of Basal Expression in Endothelial Cell Function

The Notch signaling pathway and the delta-like 4 ligand (DLL4) play key roles in embryonic vascular development. Many of the pathways involved in embryonic vascular development also play important roles in tumor angiogenesis. In this study, we assessed the expression of DLL4 in primary renal cancer and investigated the biological function of DLL4 in primary endothelial cells. Using real-time quantitative PCR and in situ hybridization, we showed that the expression of DLL4 was up-regulated within the vasculature of clear cell-renal cell carcinoma almost 9-fold more than normal kidney and was correlated with the expression of vascular endothelial growth factor (VEGF). The expression of DLL4 in endothelial cells was up-regulated by VEGF and basic fibroblast growth factor synergistically, and by hypoxia through hypoxia-inducible factor 1alpha. Down-regulation of DLL4 expression with RNA interference led to decreased expression of HEY1 and EphrinB2, and the inhibition of endothelial cell proliferation, migration, and network formation, all of which are important processes in tumor angiogenesis. The inhibition of proliferation occurred via the induction of cell cycle arrest in G0-G1 by increased expression of p21 and decreased phosphorylation of retinoblastoma. We conclude that an optimal window of the DLL4 expression is essential for tumor angiogenesis and that selective modulation of the DLL4 expression within human tumors may represent a potential novel antiangiogenic therapy.

Preoperative Chemotherapy Plus Trastuzumab, Lapatinib, or Both in Human Epidermal Growth Factor Receptor 2–Positive Operable Breast Cancer: Results of the Randomized Phase II CHER-LOB Study

PURPOSE This is a noncomparative, randomized, phase II trial of preoperative taxane-anthracycline in combination with trastuzumab, lapatinib, or combined trastuzumab plus lapatinib in patients with human epidermal growth factor receptor 2 (HER2) -positive, stage II to IIIA operable breast cancer. The primary aim was to estimate the percentage of pathologic complete response (pCR; no invasive tumor in breast and axillary nodes). PATIENTS AND METHODS In the three arms, chemotherapy consisted of weekly paclitaxel (80 mg/m(2)) for 12 weeks followed by fluorouracil, epirubicin, and cyclophosphamide for four courses every 3 weeks. The patients randomly assigned to arm A received a 4-mg loading dose of trastuzumab followed by 2 mg weekly; in arm B patients received lapatinib 1,500 mg orally (PO) daily; and in arm C, patients received trastuzumab and lapatinib 1,000 mg PO daily. RESULTS A total of 121 patients were randomly assigned. Diarrhea and dermatologic and hepatic toxicities were observed more frequently in patients receiving lapatinib. No episodes of congestive heart failure were observed. The rates of breast-conserving surgery were 66.7%, 57.9%, and 68.9% in arms A, B and C, respectively. The pCR rates were 25% (90% CI, 13.1% to 36.9%) in arm A, 26.3% (90% CI, 14.5% to 38.1%) in arm B, and 46.7% (90% CI, 34.4% to 58.9%) in arm C (exploratory P = .019). CONCLUSION The primary end point of the study was met, with a relative increase of 80% in the pCR rate achieved with chemotherapy plus trastuzumab and lapatinib compared with chemotherapy plus either trastuzumab or lapatinib. These data add further evidence supporting the superiority of a dual-HER2 inhibition for the treatment of HER2-positive breast cancer.

The Role of ATF4 Stabilization and Autophagy in Resistance of Breast Cancer Cells Treated with Bortezomib

The ubiquitin-proteasome system plays a key regulatory role in cellular homeostasis. The inhibition of the 26S proteasome by Bortezomib leads to the accumulation of misfolded proteins, resulting in endoplasmic reticulum stress followed by a coordinated cellular response called unfolded protein response (UPR). Endoplasmic reticulum stress is also a potent inducer of macroautophagy. Bortezomib is a selective and potent inhibitor of the 26S proteasome and is approved for the treatment of multiple myeloma. Clinical trials with Bortezomib have shown promising results for some types of cancers, but not for some others, including those of the breast. In this study, we show that Bortezomib induces the UPR and autophagy in MCF7 breast cancer cells. Surprisingly, Bortezomib did not induce phosphorylation of PERK, a key initial step of the UPR. We show that induction of autophagy by Bortezomib is dependent on the proteasomal stabilisation of ATF4 and up-regulation of LC3B by ATF4. We show that ATF4 and LC3B play a critical role in activating autophagy and protecting cells from Bortezomib-induced cell death. Our experiments also reveal that HDAC6 knockdown results in decreased LC3B protein and reduced autophagy. Our work shows that the induction of autophagy through ATF4 may be an important resistance mechanism to Bortezomib treatment in breast cancer, and targeting autophagy may represent a novel approach to sensitize breast cancers to Bortezomib.

Hypoxia-Inducible Factor-1α Expression Predicts a Poor Response to Primary Chemoendocrine Therapy and Disease-Free Survival in Primary Human Breast Cancer

Purpose: To investigate the relationship of hypoxia-inducible factor-1α (HIF-1α) tumor expression in predicting the response to epirubicin and disease-free survival (DFS) in patients with breast cancer enrolled in a single institution trial of primary anthracycline and tamoxifen therapy. Experimental Design: The expression of HIF-1α was assessed by immunohistochemistry in 187 patients with T2-4 N0-1 breast cancer enrolled in a randomized trial comparing four cycles of single agent epirubicin versus epirubicin + tamoxifen as primary systemic treatment. All patients postoperatively received four cycles of the four weekly i.v. CMF regimen (cyclophosphamide, methotrexate, and 5-fluorouracil). Patients with estrogen receptor (ER)-positive primary tumors also underwent 5 years of treatment with adjuvant tamoxifen. Carbonic anhydrase IX (CAIX) was also scored as a marker of HIF activity. Results: Overall response to therapy progressively decreased with increasing tumor HIF-1α (P < 0.05), and HIF-1α was an independent predictor of response (P < 0.048). HIF-1α expression was also associated with a significantly shorter DFS (P < 0.02) in all patients and in ER-positive but not in ER-negative patients. Furthermore, CAIX positivity conferred a significantly shorter DFS (P = 0.02) compared with CAIX-negative tumors in patients with HIF-1α-negative tumors. Conclusions: HIF-1α expression in patients with breast cancer is a marker of poor therapy response and outcome, especially in ER-positive patients. The combination of two hypoxia markers has greater utility than assessing just one, and patients with hypoxia markers in their tumors may be suitable for administration of drugs that reduce HIF-1α expression and increase oxygen delivery to the tumor bed before starting neoadjuvant therapies.

Epithelial-mesenchymal transition and breast cancer: Role, molecular mechanisms and clinical impact

Epithelial-mesenchymal transition (EMT) is defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. In this process, cells acquire molecular alterations that facilitate dysfunctional cell-cell adhesive interactions and junctions. These processes may promote cancer cell progression and invasion into the surrounding microenvironment. Such transformation has implications in progression of breast carcinoma to metastasis, and increasing evidences support most tumors contain a subpopulation of cells with stem-like and mesenchymal features that is resistant to chemotherapy. This review focuses on the physiological and pathological role of EMT process, its molecular related network, its putative role in the metastatic process and its implications in response/resistance to the current and/or new approaching drugs in the clinical management of breast cancer.

Breast tumour angiogenesis

The central importance of tumour neovascularization has been emphasized by clinical trials using antiangiogenic therapy in breast cancer. This review gives a background to breast tumour neovascularization in in situ and invasive breast cancer, outlines the mechanisms by which this is achieved and discusses the influence of the microenvironment, focusing on hypoxia. The regulation of angiogenesis and the antivascular agents that are used in an antiangiogenic dosing schedule, both novel and conventional, are also summarized.

Sensitization of BCL-2–expressing breast tumors to chemotherapy by the BH3 mimetic ABT-737

Overexpression of the prosurvival protein BCL-2 is common in breast cancer. Here we have explored its role as a potential therapeutic target in this disease. BCL-2, its anti-apoptotic relatives MCL-1 and BCL-XL, and the proapoptotic BH3-only ligand BIM were found to be coexpressed at relatively high levels in a substantial proportion of heterogeneous breast tumors, including clinically aggressive basal-like cancers. To determine whether the BH3 mimetic ABT-737 that neutralizes BCL-2, BCL-XL, and BCL-W had potential efficacy in targeting BCL-2–expressing basal-like triple-negative tumors, we generated a panel of primary breast tumor xenografts in immunocompromised mice and treated recipients with either ABT-737, docetaxel, or a combination. Tumor response and overall survival were significantly improved by combination therapy, but only for tumor xenografts that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitizes the tumor cells to docetaxel. Combination therapy was accompanied by a marked increase in apoptosis and dissociation of BIM from BCL-2. Notably, BH3 mimetics also appeared effective in BCL-2–expressing xenograft lines that harbored p53 mutations. Our findings provide in vivo evidence that BH3 mimetics can be used to sensitize primary breast tumors to chemotherapy and further suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer.

Sunitinib Plus Paclitaxel Versus Bevacizumab Plus Paclitaxel for First-Line Treatment of Patients With Advanced Breast Cancer: A Phase III, Randomized, Open-Label Trial

INTRODUCTION A multicenter, open-label phase III study was conducted to test whether sunitinib plus paclitaxel prolongs progression-free survival (PFS) compared with bevacizumab plus paclitaxel as first-line treatment for patients with HER2(-) advanced breast cancer. PATIENTS AND METHODS Patients with HER2(-) advanced breast cancer who were disease free for ≥ 12 months after adjuvant taxane treatment were randomized (1:1; planned enrollment 740 patients) to receive intravenous (I.V.) paclitaxel 90 mg/m(2) every week for 3 weeks in 4-week cycles plus either sunitinib 25 to 37.5 mg every day or bevacizumab 10 mg/kg I.V. every 2 weeks. [corrected] RESULTS The trial was terminated early because of futility in reaching the primary endpoint as determined by the independent data monitoring committee during an interim futility analysis. At data cutoff, 242 patients had been randomized to sunitinib-paclitaxel and 243 patients to bevacizumab-paclitaxel. Median PFS was shorter with sunitinib-paclitaxel (7.4 vs. 9.2 months; hazard ratio [HR] 1.63 [95% confidence interval (CI), 1.18-2.25]; 1-sided P = .999). At a median follow-up of 8.1 months, with 79% of sunitinib-paclitaxel and 87% of bevacizumab-paclitaxel patients alive, overall survival analysis favored bevacizumab-paclitaxel (HR 1.82 [95% CI, 1.16-2.86]; 1-sided P = .996). The objective response rate was 32% in both arms, but median duration of response was shorter with sunitinib-paclitaxel (6.3 vs. 14.8 months). Bevacizumab-paclitaxel was better tolerated than sunitinib-paclitaxel. This was primarily due to a high frequency of grade 3/4, treatment-related neutropenia with sunitinib-paclitaxel (52%) precluding delivery of the prescribed doses of both drugs. CONCLUSION The sunitinib-paclitaxel regimen evaluated in this study was clinically inferior to the bevacizumab-paclitaxel regimen and is not a recommended treatment option for patients with advanced breast cancer.

Phosphorylated ERα, HIF-1α, and MAPK Signaling As Predictors of Primary Endocrine Treatment Response and Resistance in Patients With Breast Cancer

PURPOSE We aimed to identify signaling pathways involved in the response and resistance to aromatase inhibitor therapy in patients with breast cancer. PATIENTS AND METHODS One hundred fourteen women with T2-4 N0-1, estrogen receptor (ER) alpha-positive tumors were randomly assigned to neoadjuvant letrozole or letrozole plus metronomic cyclophosphamide. Twenty-four tumor proteins involved in apoptosis, cell survival, hypoxia, angiogenesis, growth factor, and hormone signaling were assessed by immunohistochemistry in pretreatment samples (eg, caspase 3, phospho- mammalian target of rapamycin, hypoxia-inducible factor 1alpha [HIF-1alpha], vascular endothelial growth factor, mitogen-activated protein kinase [MAPK], phosphorylated epidermal growth factor receptor, phosphorylated ERalpha [pERalpha]). A multivariate generalized linear regression approach was applied using a penalized least-square minimization to perform variable selection and regularization. Ten-fold cross-validation and iterative leave-one-out were employed to validate and test the model, respectively. Tumor size, nodal status, age, tumor grade, histological type, and treatment were included in the analysis. RESULTS Ninety-one patients (81%) attained a disease response, 48 achieved a complete clinical response (43%) whereas 22 did not respond (19%). Increased pERalpha and decreased p44/42 MAPK were significant factors for complete response to treatment in all leave-one-out iterations. Increased p44/42 MAPK and HIF-1alpha were significant factors for treatment resistance in all leave-one-out iterations. There was no significant interaction between these variables and treatment. CONCLUSION Activated ERalpha form was an independent factor for sensitivity to chemoendocrine treatment, whereas HIF-1alpha and p44/42 MAPK were independent factors for resistance. Although further confirmatory analyses are needed, these findings have clear potential implications for future strategies in the management of clinical trials with aromatase inhibitors in the breast cancer.

Immunomodulation of FOXP3+ Regulatory T Cells by the Aromatase Inhibitor Letrozole in Breast Cancer Patients

Purpose: We have shown previously that tumor infiltration by FOXP3+ regulatory T cells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulates Treg numbers in mice and promotes the proliferation of human Tregs, we hypothesized that blocking estrogen receptor-α signaling would abrogate Tregs and be associated with response to hormonal therapy and increased survival. Experimental Design: FOXP3+ Tregs were quantified in tumor samples collected at baseline by incisional biopsy and after 6 months at definitive surgery in 83 elderly breast cancer patients (T2-4 N0-1) enrolled in a randomized phase II trial based on 6 months of primary letrozole (2.5 mg/d) or 6 months of letrozole plus oral “metronomic” cyclophosphamide (50 mg/d). Results: Treg number ranged from 0 to 380 (median, 30) before treatment and from 0 to 300 (median, 8) after treatment. There was a significant reduction in Tregs in letrozole and letrozole-cyclophosphamide patients (P < 0.0001 and P < 0.002, respectively) after treatment. Treg number at residual histology was inversely related with response (P < 0.03 and P = 0.50, respectively) and a greater Treg reduction was observed in responding patients (P < 0.03). Conclusion: This study suggests that aromatase inhibitors may have an indirect antitumor mechanism of action through reducing Tregs in breast tumors and may be of use in estrogen receptor-α-negative tumors in combination with immunotherapy approaches.