L. Damstrup

Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials

The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intracellular region of the receptor. This tyrosine kinase phosphorylates a number of intracellular substrates that activates pathways leading to cell growth, DNA synthesis and the expression of oncogenes such as fos and jun. EGFR is thought to be involved the development of cancer, as the EGFR gene is often amplified, and/or mutated in cancer cells. In this review we will focus on: (I) the structure and function of EGFR, (II) implications of receptor/ligand coexpression and EGFR mutations or overexpression, (III) its effect on cancer cells, (IV) the development of the malignant phenotype and (V) the clinical aspects of therapeutic targeting of EGFR.

The type III epidermal growth factor receptor mutation Biological significance and potential target for anti-cancer therapy

Mutations in the epidermal growth factor receptor occur frequently in a number of human tumours including gliomas, non-small-cell lung carcinomas, ovarian carcinomas and prostate carcinomas. The type III epidermal growth factor receptor mutation (variously named EGFRvIII, de2-7 EGFR or AEGFR), which lacks a portion of the extracellular ligand binding domain, is the most common. Here, we review the current status with regard to the role of EGFRvIII in human cancers. A detailed discussion of the formation of EGFRvIII and its structure at the protein level are likewise included along with a discussion of its more functional roles. The design and use (preclinical and clinical) of small molecule inhibitors, antibodies, and antisense oligonucleotides against wild-type EGFR are considered in detail as these strategies can be directly adapted to target EGFRvIII. Finally, the status of EGFRvIII targeted therapy is reviewed.

Analysis of the epidermal growth factor receptor specific transcriptome: Effect of receptor expression level and an activating mutation

Overexpression or expression of activating mutations of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. The present study employed Affymetrix® oligonucleotide arrays to profile genes induced by ligand‐activated EGFR with the receptor either moderately expressed or overexpressed at an in‐itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes encoding proteins with functions in promoting cell proliferation, invasion, antiapoptosis, and angiogenesis featured prominently in the EGFRvIII‐ and EGFR‐expressing cells. Surprisingly, it was found that ligand‐activated EGFR induced the expression of a large group of genes known to be inducible by interferons. Expression of this module was absent in the EGFRvIII‐expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor‐mediated events. Furthermore, activation of this module correlated with activation of STAT1 and STAT3. The results thus demonstrate that ligand‐activated EGFR at different expression levels results in different kinetics of signaling and induction of gene expression. In addition, the constitutively active variant EGFRvIII seems to activate only a subset of signal pathways and induce a subset of genes as compared to the ligand‐activated EGFR.

Growth suppression by transforming growth factor beta 1 of human small-cell lung cancer cell lines is associated with expression of the type II receptor.

Nine human small-cell lung cancer cell lines were treated with transforming growth factor beta 1 (TGF-beta 1). Seven of the cell lines expressed receptors for transforming growth factor beta (TGF-beta-r) in different combinations between the three human subtypes I, II and III, and two were receptor negative. Growth suppression was induced by TGF-beta 1 exclusively in the five cell lines expressing the type II receptor. For the first time growth suppression by TGF-beta 1 of a cell line expressing the type II receptor without coexpression of the type I receptor is reported. No effect on growth was observed in two cell lines expressing only type III receptor and in TGF-beta-r negative cell lines. In two cell lines expressing all three receptor types, growth suppression was accompanied by morphological changes. To evaluate the possible involvement of the retinoblastoma protein (pRb) in mediating the growth-suppressive effect of TGF-beta 1, the expression of functional pRb, as characterised by nuclear localisation, was examined by immunocytochemistry. Nuclear association of pRb was only seen in two of the five TGF-beta 1-responsive cell lines. These results indicate that in SCLC pRb is not required for mediation of TGF-beta 1-induced growth suppression.

Epidermal growth factor receptor mutation type III transfected into a small cell lung cancer cell line is predominantly localized at the cell surface and enhances the malignant phenotype.

In the present study we transfected the epidermal growth factor receptor (EGFR)‐negative small cell lung cancer cell line, GLC3, with the type III EGFR mutation (EGFRvIII). The EGFRvIII protein could be detected by Western blot analysis as a 145‐kDa protein, which by immunohistochemistry appeared to be localized at the cell surface. Ultrastructurally EGFRvIII was expressed mainly at the cell surface with clusters at cell–cell contacts. In the in vitro invasion assay, GLC3‐EGFRvIII cells had a ≈5‐fold increased invasion compared with uninduced GLC3‐EGFRvIII, GLC3‐Tet‐On and the parental cell line. GLC3‐Tet‐On appeared uniform in size with adherence junctions at cell–cell contacts. In uninduced GLC3‐EGFRvIII cells adherence junctions were also present but less distinct. In doxycycline‐pretreated GLC3‐EGFRvIII cells, adherence junctions were absent. We conclude that the expression of EGFRvIII results in a more malignant phenotype. This effect appears to involve the disruption of adherence junctions.

Phase II activity of belinostat (PXD-101), carboplatin, and paclitaxel in women with previously treated ovarian cancer.

Background Preclinical data show that belinostat (Bel) is synergistic with carboplatin and paclitaxel in ovarian cancer. To further evaluate the clinical activity of belinostat, carboplatin, and paclitaxel (BelCaP), a phase 1b/2 study was performed, with an exploratory phase 2 expansion planned specifically for women with recurrent epithelial ovarian cancer (EOC). Methods Thirty-five women were treated on the phase 2 expansion cohort. BelCap was given as follows: belinostat, 1000 mg/m2 daily for 5 days with carboplatin, AUC 5; and paclitaxel, 175 mg/m2 given on day 3 of a 21-day cycle. The primary end point was overall response rate (ORR), using a Simon 2 stage design. Results The median age was 60 years (range, 39–80 years), and patients had received a median of 3 prior regimens (range, 1–4). Fifty-four percent had received more than two prior platinum-based combinations, sixteen patients (46%) had primary platinum-resistant disease, whereas 19 patients (54%) recurred within 6 months of their most recent platinum treatment. The median number of cycles of BelCaP administered was 6 (range, 1–23). Three patients had a complete response, and 12 had a partial response, for an ORR of 43% (95% confidence interval, 26%–61%). When stratified by primary platinum status, the ORR was 44% among resistant patients and 63% among sensitive patients. The most common drug-related adverse events related to BelCaP were nausea (83%), fatigue (74%), vomiting (63%), alopecia (57%), and diarrhea (37%). With a median follow-up of 4 months (range, 0–23.3 months), 6-month progression-free survival is 48% (95% confidence interval, 31%–66%). Median overall survival was not reached during study follow-up. Conclusions Belinostat, carboplatin, and paclitaxel combined was reasonably well tolerated and demonstrated clinical benefit in heavily-pretreated patients with EOC. The addition of belinostat to this platinum-based regimen represents a novel approach to EOC therapy and warrants further exploration.

In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines.

Review of the curative role of radiotherapy in the treatment of non-small cell lung cancer

The present paper is a comprehensive review of available data concerning the role of radiotherapy as an intended curative treatment in patients with non-small cell lung cancer (NSCL). The following issues are reviewed (1) optimal dose, (2) optimal fractionation, (3) optimal treatment planning, (4) clinical results in terms of single treatment and combined treatment with either surgery or chemotherapy. In resectable NSCLC high dose radiotherapy to small localized tumours gives a 5-year survival rate of 7-38%. It is concluded that this treatment modality is appropriate for certain selected patients who refuse to have surgery, who have medical contradications for surgery, or who are of old age. It is discussed whether the treatment should be split course, continuous, hypo-og hyperfraction. A total dose of 55 Gy must be given. CT scanning should be mandatory for optimal planning and therapy. The literature does not give a conclusive answer to whether preoperative or postoperative radiotherapy is indicated. The data indicate that patients with Stage III NSCLC will benefit from a combined treatment modality in terms of chemotherapy based on high dose cisplatinum and radiotherapy. The main conclusion of the review is that many areas with randomized controlled trials are needed in order to answer the critical issue of the role of radiotherapy in the treatment of NSCLS.

Gene delivery by an epidermal growth factor/DNA polyplex to small cell lung cancer cell lines expressing low levels of epidermal growth factor receptor.

In the present study, we wanted to determine whether efficient gene delivery using an epidermal growth factor (EGF)/DNA polyplex could be accomplished in small cell lung cancer (SCLC) cell lines expressing low EGF receptor (EGFR) levels. EGFR expression levels and transduction efficiencies with polyplexes were examined in five SCLC cell lines and two controls. EGFR expression was examined by binding assays and demonstrated low EGFR levels ranging from 3.6 to 87.4 fmol/mg protein. The SCLC cell lines exhibited high sensitivity to adenovirus infection, which was an important determinant for transduction efficiency when adenovirus was used as an endosomolytic agent. The transduction efficiencies with EGF/DNA polyplexes ranged from 41% ± 3.5% to 73% ± 4.6% in the EGFR-positive SCLC cell lines. In the controls lacking EGFRs, only 5% ± 1.0% and 8% ± 1.8% of the cells were transduced. Furthermore, the transduction efficiency could be reduced from 50% ± 4.9% to 18% ± 1.1% when excess EGF was added to compete with the EGF/DNA polyplexes. In the present study, receptor-targeted gene delivery to SCLC cell lines has been demonstrated for the first time. Our results indicate that even low receptor expression levels in the target cells are sufficient for efficient and specific in vitro gene delivery with EGF/DNA polyplexes.

Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line

Previous studies have shown a correlation between expression of the EGF receptor type III mutation (EGFRvIII) and a more malignant phenotype of various cancers including: non-small-cell lung cancer, glioblastoma multiforme, prostate cancer and breast cancer. Thus, a detailed molecular genetic understanding of how the EGFRvIII contributes to the malignant phenotype is of major importance for future therapy. The GeneChip Hu6800Set developed by Affymetrix was used to identify changes in gene expression caused by the expression of EGFRvIII. The cell line selected for the study was an EGF receptor negative small-cell-lung cancer cell line, GLC3, stably transfected with the EGFRvIII gene in a Tet-On system. By comparison of mRNA levels in EGFRvIII-GLC3 with those of Tet-On-GLC3, it was found that the levels of mRNAs encoding several transcription factors (ATF-3, JunD, and c-Myb), cell adhesion molecules (CD36, CD24), signal transduction related molecules (MKP-1) and other molecules related to cancer (CD98, thymosin beta-10) were altered in the EGFRvIII transfected cell line. Northern hybridisations and Western blot analyses were used to verify selected results. The results indicate that expression of EGFRvIII alters expression of genes involved in the control of cell growth, survival and motility.