Intrinsic or acquired resistance to anticancer agents is a major obstacle to the success of chemotherapy. In the present work, we compared the invasive properties of drug sensitive NB with their drug-resistant sublines, as well as intrinsically resistant NB which were further treated with VCR or DOX. The selection of the various tumor sublines mimicked the severe problems arising in patients during chemotherapy. Based on our model, we demonstrate that drug resistant cancer cells develop an increased malignant phenotype, in part due to enhanced adhesion and transendothelial penetration, accompanied by a significant down-regulation of the adhesion receptor NCAM.
Our findings might explain the paradoxical situation that tumors sometimes become progressively more malignant during the course of chemotherapeutic treatment. Slotman et al. found an increased incidence of hematogenous metastases with induction chemotherapy for patients with advanced head-and-neck squamous carcinomas, compared to patients receiving only surgery and radiotherapy. They also found an increase in metastases at sites other than the usual pulmonary site [16
]. Stefani and coworkers reported that the use of hydroxyurea in combination with radiotherapy for the treatment of head and-neck cancer yielded no benefit in terms of response rate or survival, but rather increased the incidence of distant metastases from 8% to 23% [17
]. The administration of a single drug dose may be sufficient to induce spontaneous metastasis [18
Two reports have been published recently which underline our observations and suggest that drug resistance in melanoma and carcinoma cells may confer to a more invasive phenotype [19
]. Nevertheless, it is still hypothesized that resistance to cytotoxic agents and invasive potential is associated with the over-expression of Pgp drug transporters [19
]. Notably, increased Pgp expression was found in metastatic NB cells from bone marrow samples, as compared to non-metastatic NB [22
]. Previous experiments on UKF-NB-2 and UKF-NB-3 cells also revealed high amounts of functionally active Pgp in both DOX and VCR resistant sublines. However, expression of Pgp was 3.5-fold higher in UKF-NB-2DOX
when compared to UKF-NB-2VCR
cells, and therefore does not correlate to the changes seen with respect to cell adhesion and transendothelial penetration (UKF-NB-2DOX
). Furthermore, pharmacological blockage of Pgp in resistant UKF-NB-2 and UKF-NB-3 cell lines restored chemosensitivity, but had no effect on colony formation and cell survival under serum free conditions [7
Therefore, up-regulation of Pgp itself probably does not play a leading role in increasing NB malignant biology. However, concomitant deregulation of other pathways may result in increased metastatic potential, thus leading to a poorer prognosis of advanced forms of NB disease. In our cell culture model, modulation of NCAM seems to be the major parameter responsible for altered cell adhesion and penetration. This assumption is based on three observations: 1. Enhanced tumor binding and transmigration of drug resistant cell populations only occured when NCAM receptor expression was reduced. 2. NCAM loss strongly correlated with the elevation of adhesion and penetration capacity, and 3. Transfection of UKF-NB-4VCR
cells with NCAM cDNA up-regulated the NCAM expression level and diminished the interaction events between tumor and endothelial cells. In good accordance to the present data, we recently demonstrated an inverse correlation between NCAM expression and NB cell adhesion, assessed on 11 NB cell lines. In particular, transfection with a cDNA encoding the human NCAM-140 kD isoform enhanced NCAM expression and diminished initial NB cell adhesion, treatment with NCAM antisense oligonucleotides reduced NCAM surface level and induced up-regulation of NB cell adhesion to endothelium [10
Several studies have demonstrated the important role of NCAM in tumor migration and metastasis, with an inverse relationship between migratory potential of the cells and NCAM expression. In primitive neuroectodermal tumor cells, an increase in NCAM was paralleled by a significant reduction in cellular motility and adhesion capacity [23
]. In a rat model, NCAM-transfected glioma tumor cells became less invasive and destructive than control cells with a low NCAM expression level [25
]. Diminished expression of NCAM was also associated with clinically aggressive colon cancers [26
], and dissemination of pancreatic beta tumor cells [29
]. Tezel et al. suggested that NCAM expression in tubular adenocarcinoma of the pancreas has a significant impact on overall patient survival [31
]. It is currently assumed that NCAM, in its function as a homophilic receptor, stabilizes the primary tumor or tumor cell aggregates, while circulating in the blood vessels. Reduction of the NCAM expression level might lead to a reduction in cell-cell binding forces, and hence to the release of tumors as single cells. The less NCAM, the more metastatic cells leave the tumor mass, and the more penetration events can take place [10
]. Consequently, NCAM loss observed in DOX and VCR resistant NB tumor cells allows more cells to transmigrate the endothelial blood barrier and extravasate into surrounding tissue. In line with this, establishment of DOX or VCR resistant human glioma cells was accompanied by NCAM reduction and a concomitant down-regulation of adhesiveness [32
]. Analyses of tumor samples from relapsed patients have to be carried out to strenghthen the relevance of the in vitro findings.
Recently, it has been documented that the loss of NCAM function causes the formation of lymph node metastasis in pancreatic beta cell carcinogenesis via vascular endothelial growth factor -C and -D-mediated lymphangiogenesis [33
]. In a previous work, loss of NCAM resulted in a failure to activate beta1 integrins via fibroblast growth factor receptor signalling [29
]. Both findings implicate that NCAM, beside its role as an aggregation stabilizer, may also act in terms of a "metastasis suppressor protein", controlling proteins and genes that specifically inhibit the ability of tumor cells from forming metastases [34
Our hypothesis of drug evoked alterations of NB tumor cell binding and transvasation also appears to be applicable to tumor cells with intrinsic drug resistance. UKF–NB-4 cells with a multidrug resistant phenotype retained the potential to become even more aggressive when treated with DOX or VCR. From a clinical viewpoint, chemotherapy of NB might induce adverse effects concerning tumor biology. In fact, single chemotherapy may amplify the process of tumor progression if drug-resistant tumor subclones are present in the initial tumor burden.
The finding that extended drug exposure contributes to distinct modifications of adhesion receptors and invasive potential of single cancer cells, independent of Pgp over-expression, may be a signal to re-assess current therapeutic protocols. In fact, the use of escalating doses of cyclophosphamide and prolonging conventional chemotherapy with the same drugs has failed to improve the metastasis complete response rate in NB patients [35
]. Since initial pharmacological attempts to inhibit drug efflux and increase intracellular drug concentrations have not provided the desired clinical benefit in relapsed or resistant paediatric cancers [36
], the consequences of drug resistance might preferably be treated by agents which reverse cellular adhesion. In this context, the branched-chain fatty acid valproate has been demonstrated to inhibit tumor cell motility and adhesion by up-regulating NCAM [37
]. The same is true for the differentiation inducing compound trichostatin A [39
]. A significant increase of NCAM expression level accompanied by blocking cellular adhesion has also been observed when neuroblastoma cells were treated with retinoic acid [40
]. The introduction of differentiation inducing drugs may therefore advance the treatment of relapsed tumors. Currently, several studies dealing with this issue are underway.
It is important to note that extended drug exposure evoked different responses in the in vitro system described here. Although drug resistance was established in all tumor sublines, VCR resistance led to increased attachment of UKF-NB-2, UKF-NB-3 or UKF-NB-4 cell lines, but not of IMR-32, whereas DOX resistance induced marked elevation of IMR-32 attachment but had no influence on UKF-NB-3 adhesion. Alterations in the NCAM expression level followed the same pattern. Therefore, modifications of the invasive program cannot be expected in every case where drug resistance in tumor cells develops. VCR or DOX resistance might convert NCAM triggered cell adhesion in some but not in all tumor subpopulations, or in some but not in all tumor patients where chemotherapy has failed. It is still unknown how drug resistant tumor cells are selected to became "responders", i.e. increase their invasive activity. However, if our hypothesis holds true, a critical selection of those "responding" patients should be carried out who might be predestined for innovative antitumoral, adhesion blocking strategies.