Marianna Gallo

The role of the EGFR signaling in tumor microenvironment

The epidermal growth factor receptor (EGFR) family comprehends four different tyrosine kinases (EGFR, ErbB‐2, ErbB‐3, and ErbB‐4) that are activated following binding to epidermal growth factor (EGF)‐like growth factors. It has been long established that the EGFR system is involved in tumorigenesis. These proteins are frequently expressed in human carcinomas and support proliferation and survival of cancer cells. However, activation of the EGFR in non‐malignant cell populations of the neoplastic microenvironment might also play an important role in cancer progression. EGFR signaling regulates in tumor cells the synthesis and secretion of several different angiogenic growth factors, including vascular endothelial growth factor (VEGF), interleukin‐8 (IL‐8), and basic fibroblast growth factor (bFGF). Overexpression of ErbB‐2 also leads to increased expression of angiogenic growth factors, whereas treatment with anti‐EGFR or anti‐ErbB‐2 agents produces a significant reduction of the synthesis of these proteins by cancer cells. EGFR expression and function in tumor‐associated endothelial cells has also been described. Therefore, EGFR signaling might regulate angiogenesis both directly and indirectly. In addition, activation of EGFR is involved in the pathogenesis of bone metastases. Within the bone marrow microenvironment, cancer cells stimulate the synthesis of osteoclastogenic factors by residing stromal cells, a phenomenon that leads to bone destruction. It has been shown that EGFR signaling regulates the ability of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation. Taken together, these findings suggest that the EGFR system is an important mediator, within the tumor microenvironment, of autocrine and paracrine circuits that result in enhanced tumor growth. J. Cell. Physiol. 214: 559–567, 2008.

Mesenchymal stem cell‐derived interleukin‐6 and vascular endothelial growth factor promote breast cancer cell migration

Several different cytokines and growth factors secreted by mesenchymal stem cells (MSCs) have been hypothesized to play a role in breast cancer progression. By using a small panel of breast cancer cell lines (MCF‐7, T47D, and SK‐Br‐3 cells), we analyzed the role of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF) in the cross‐talk between MSCs and breast cancer cells. We performed migration assays in which breast cancer cells were allowed to migrate in response to conditioned medium from MSCs (MSCs‐CM), in absence or in presence of the anti‐VEGF antibody bevacizumab or an anti‐IL‐6 antibody, alone or in combination. We found that anti‐VEGF and anti‐IL‐6 antibodies inhibited the migration of breast cancer cells and that the combination had an higher inhibitory effect. We next evaluated the effects of recombinant VEGF and IL‐6 proteins on breast cancer cell growth and migration. IL‐6 and VEGF had not significant effects on the proliferation of breast carcinoma cells. In contrast, both VEGF and IL‐6 significantly increased the ability to migrate of MCF‐7, T47D and SK‐Br‐3 cells, with the combination showing a greater effect as compared with treatment with a single protein. The combination of VEGF and IL‐6 produced in breast cancer cells a more significant and more persistent activation of MAPK, AKT, and p38MAPK intracellular signaling pathways. These results suggest that MSC‐secreted IL‐6 and VEGF may act as paracrine factors to sustain breast cancer cell migration. J. Cell. Biochem. 113: 3363–3370, 2012.

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells.

Increasing evidence suggests that bone marrow‐derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor‐associated neo‐angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF‐α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF‐α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF‐α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF‐α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin‐2, granulocyte‐colony stimulating factor, hepatocyte growth factor, interleukin (IL)‐6, IL‐8, and platelet‐derived growth factor‐BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF‐α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF‐α increased the ability of MSCs to induce migration of MCF‐7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo‐angiogenesis and tumor cell migration. J. Cell. Physiol. 226: 2131–2138, 2011.

Target-based therapies in breast cancer: current status and future perspectives

Identification of molecular alterations in key proteins involved in breast cancer cell proliferation and survival resulted in the development of a new treatment strategy with target-based agents. The anti-ErbB-2 monoclonal antibody (mAb) trastuzumab and the dual epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor lapatinib are effective in patients with breast cancer that overexpresses ErbB-2. The anti-vascular endothelial growth factor-A mAb bevacizumab is approved in combination with taxanes for treatment of unselected patients with metastatic breast cancer. In addition, preclinical data suggest that signaling inhibitors can prevent or overcome resistance to endocrine therapy in estrogen receptor positive (ER+) breast cancer. However, the majority of signaling inhibitors explored in breast cancer patients has shown little activity, at least when used as monotherapy; and the results of clinical trials in ER+ breast cancer of combinations of signaling inhibitors and endocrine therapies are rather disappointing. Negative findings are likely due to mechanisms of intrinsic or acquired resistance to target-based agents. Breast carcinoma is a complex and heterogeneous disease and several different molecular alterations are involved in its pathogenesis and progression. The redundancy of oncogenic pathways activated in cancer cells, the heterogeneity of the mechanisms of resistance, and the plasticity of tumor cells that are capable to adapt to different growth conditions, significantly hamper the efficacy of each signaling inhibitor in breast cancer. Therefore, a comprehensive approach that takes into account the complexity of the disease is definitely required to improve the efficacy of target-based therapy in breast cancer.

Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells

Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27kip1 and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27kip1, with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27kip1 mRNA and in the nuclear levels of the p27kip1 transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27kip1 mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27kip1 mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27kip1 expression.

Breast cancer cells with acquired resistance to the EGFR tyrosine kinase inhibitor gefitinib show persistent activation of MAPK signaling

Although the epidermal growth factor receptor (EGFR) is frequently expressed in human primary breast carcinoma, the majority of breast cancer patients do not respond to treatment with EGFR tyrosine-kinase inhibitors such as gefitinib. We isolated through a stepwise dose escalation of the drug two gefitinib-resistant SK-Br-3 clones, ZD6 and ZD10 (ZD) cells, which showed, respectively, a three- to five-fold increase in the IC50 for gefitinib as compared with parental cells. The levels of expression of EGFR were increased in ZD cells as compared with wild-type SK-Br-3 cells. The phosphorylation of EGFR, ErbB-2, ErbB-3 and Akt was significantly reduced in gefitinib-resistant cells. In contrast, ZD cells showed levels of MAPK phosphorylation similar to untreated wild-type cells when cultured in presence of gefitinib. Persistent activation of MAPK was also observed in gefitinib-resistant clones isolated from MDA-MB-175 and MDA-MB-361 breast cancer cell lines. ZD cells showed an increased sensitivity to the MEK inhibitor PD98059 as compared with SK-Br-3 cells, and a synergistic anti-tumor effect was observed when ZD cells were treated with a combination of gefitinib and PD98059. Overexpression of a constitutively activated form of p42-MAPK in SK-Br-3 cells resulted in an approximately 50% increase in the IC50 to gefitinib. Finally, culture of ZD10 resistant cells in absence of gefitinib led to reversion of the resistant phenotype. These observations suggest that MAPK signaling might play a role in the resistance that develops in breast cancer cells after long-term exposure to gefitinib.

Src and CXCR4 are involved in the invasiveness of breast cancer cells with acquired resistance to lapatinib

Lapatinib is a dual EGFR and ErbB-2 tyrosine kinase inhibitor that has significantly improved the clinical outcome of ErbB-2-overexpressing breast cancer patients. However, patients inexorably develop mechanisms of resistance that limit the efficacy of the drug. In order to identify potential targets for therapeutic intervention in lapatinib-resistant patients, we isolated, from ErbB-2-overexpressing SK-Br-3 breast cancer cells, the SK-Br-3 Lap-R-resistant subclone, which is able to routinely grow in 1 µM lapatinib. Resistant cells have a more aggressive phenotype compared with parental cells, as they show a higher ability to invade through a matrigel-coated membrane. Lapatinib-resistant cells have an increased Src kinase activity and persistent levels of activation of ERK1/2 and AKT compared with parental cells. Treatment with the Src inhibitor saracatinib in combination with lapatinib reduces AKT and ERK1/2 phosphorylation and restores the sensitivity of resistant cells to lapatinib. SK-Br-3 Lap-R cells also show levels of expression of CXCR4 that are higher compared with parental cells and are not affected by Src inhibition. Treatment with saracatinib or a specific CXCR4 antibody reduces the invasive ability of SK-Br-3 Lap-R cells, with the two drugs showing cooperative effects. Finally, blockade of Src signaling significantly increases TRAIL-induced cell death in SK-Br-3 Lap-R cells. Taken together, our results demonstrate that breast cancer cells with acquired resistance to lapatinib have a more aggressive phenotype compared with their parental counterpart, and that Src signaling and CXCR4 play an important role in this phenomenon, thus representing potential targets for therapeutic intervention in lapatinib-resistant breast cancer patients.

A live biopsy in a small-cell lung cancer patient by detection of circulating tumor cells

A 71-year-old patient with a pulmonary lesion was diagnosed with a low-grade neuroendocrine tumor following examination of a fine needle aspiration biopsy. Analysis of a peripheral blood sample with the CellSearch system revealed the presence of putative circulating tumor cells (CTC) that were positive for EpCAM and cytokeratin (CK) expression. Since EpCAM is not usually expressed in neuroendocrine tumors, we performed a biopsy of liver metastases. Morphological and immunophenotypical characterization revealed that the patient had an EpCAM and CK positive small-cell lung cancer (SCLC). By using the CellSearch apparatus, EpCAM/CK positive CTC were detected in peripheral blood samples from 3 out of 4 additional SCLC patients. This study is the first to demonstrate that CTC can be identified in SCLC patients by using the CellSearch system.

EGFR and MEK Blockade in Triple Negative Breast Cancer Cells.

Although evidence suggests that the RAF/MEK/ERK pathway plays an important role in triple negative breast cancer (TNBC), resistance to MEK inhibitors has been observed in TNBC cells. Different mechanisms have been hypothesized to be involved in this phenomenon, including receptor tyrosine kinase‐dependent activation of the PI3K/AKT pathway. In this study, we analyzed the effects of the MEK1/2 inhibitor selumetinib in combination with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in a panel of TNBC cell lines that showed different levels of sensitivity to single‐agent selumetinib: SUM‐149 and MDA‐MB‐231 cells resulted to be sensitive, whereas SUM‐159, MDA‐MB‐468 and HCC70 cells were relatively resistant to the drug. Treatment of TNBC cells with selumetinib produced an increase of the phosphorylation of the EGFR both in selumetinib‐sensitive SUM‐149, MDA‐MB‐231 and in selumetinib‐resistant MDA‐MB‐468 TNBC cells. The combination of selumetinib and gefitinib resulted in a synergistic growth inhibitory effect in all the TNBC cell lines, although the IC50 was not reached in SUM‐159 and MDA‐MB‐468 cells. This effect was associated with an almost complete suppression of ERK1/2 activation and a reduction of selumetinib‐induced AKT phosphorylation. In addition, in selumetinib‐sensitive TNBC cells the combination of selumetinib and gefitinib induced a significant G0/G1 cell cycle arrest and apoptosis. Taken together, our data demonstrated that blockade of the EGFR might efficiently increase the antitumor activity of selumetinib in a subgroup of TNBC and that this phenomenon might be related to the effects of such combination on both ERK1/2 and AKT activation. J. Cell. Biochem. 116: 2778–2785, 2015.

Pharmacokinetic evaluation of zoledronic acid

Introduction: Increased bone resorption is associated with several diseases, including osteoporosis, bone metastases and Pagets disease of bone. Zoledronic acid (ZA) is the most potent of the clinically available bisphosphonates. In addition to its antiresorptive activity, there has been increasing evidence to suggest that it also has anticancer properties. Areas covered: This article is complied through PubMed and Medline databases searches on ZA. In this review, the authors summarize the current knowledge (up to December 2010) on the pharmacodynamic and pharmacokinetic properties of ZA. Expert opinion: ZA is a well-tolerated and effective drug in the management of metabolic as well as cancer-related bone disease. Clinical benefits in cancer patients include improvement in bone pain, reduction in skeletal events and delay of time to first skeletal event. However, novel indications for this drug are emerging from clinical studies in early breast cancer. Recent findings suggest that the addition of ZA to endocrine therapy can significantly prevent bone loss in premenopausal patients. Increasing evidence also indicates a potential anticancer activity of ZA, although this property needs to be further explored.