Hannie Sietsma

Frequent and Focal FGFR1 Amplification Associates with Therapeutically Tractable FGFR1 Dependency in Squamous Cell Lung Cancer

FGFR1 amplification provides a therapeutic target for squamous cell lung cancer, which is resistant to other targeted lung cancer drugs. A Smoking Gun for Lung Cancer Detectives and scientists alike need strong evidence to take their cases to the judge, who for scientists is often a patient with a deadly disease. Yet, new culprits are sometimes found that can break a case wide open. Lung cancer, which accounts for more than 10% of the global cancer burden, has a poor prognosis and inadequately responds to chemotherapy and radiotherapy. New targeted treatments for lung adenocarcinomas inhibit the oncogenic versions of signaling protein kinases that arise from mutations typically found in lung cancer patients who have never smoked. However, smokers frequently suffer from a different deviant, squamous cell lung cancers, for which there are no known molecular genetic targets for therapy. Now, Weiss et al. have fingered a new suspect in smoking-related lung cancer: amplification of the FGFR1 gene, which encodes the fibroblast growth factor receptor 1 tyrosine kinase (FGFR1). To identify therapeutically viable genetic alterations that may influence squamous cell lung cancer, Weiss et al. performed genomic profiles on a large set of lung cancer specimens. Squamous cell lung cancer samples showed FGFR1 amplification, which was not found in other lung cancer subtypes. The authors then determined that a molecule that broadly inhibits FGF receptor function could block tumor growth and cause cell death in the cancers that expressed high amounts of the FGFR1 gene product in a manner that was dependent on FGFR1 expression. Moreover, FGFR1 inhibition resulted in a considerable decrease in tumor size in a mouse model of FGFR1-amplified lung cancer. This culmination of evidence implies that inhibition of this receptor tyrosine kinase should be explored as a candidate therapy for corralling squamous cell lung cancer in smokers. Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1V561M), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.

Mutations in the DDR2 Kinase Gene Identify a Novel Therapeutic Target in Squamous Cell Lung Cancer

UNLABELLED While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials. SIGNIFICANCE DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs.

Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

Multidrug‐resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug‐resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug‐resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell‐free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.

Randomized, Placebo-Controlled Phase III Study of Docetaxel Plus Carboplatin With Celecoxib and Cyclooxygenase-2 Expression As a Biomarker for Patients With Advanced Non–Small-Cell Lung Cancer: The NVALT-4 Study

PURPOSE Cyclooxygenase-2 (COX-2) protein expression in patients with non-small-cell lung cancer (NSCLC) may be not only a prognostic marker but also predictive for COX-2 inhibition. We hypothesized that COX-2 expression is associated with shorter survival and that celecoxib, being a potent COX-2 inhibitor, increases tumor response and survival. PATIENTS AND METHODS A phase III study was performed in patients with stage IIIb/IV NSCLC who had pathologic confirmation, no prior chemotherapy, Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate organ function. Treatment consisted of docetaxel and carboplatin every 3 weeks for five cycles. Patients were randomly assigned to receive celecoxib 400 mg or placebo twice daily. COX-2 expression on tumor cells was detected by immunohistochemistry. Primary end point was overall survival (OS). RESULTS From July 2003 to December 2007, 561 patients were randomly assigned. Toxicity was mild, and no increase in cardiovascular events was observed. Tumor response was 38% in the celecoxib arm and 30% in the placebo arm (P = .08). Median progression-free survival was 4.5 months (95% CI, 4.0 to 4.8) for the celecoxib arm and 4.0 months (95% CI, 3.6 to 4.9) for the placebo arm (hazard ratio [HR], 0.8; 95% CI, 0.6 to 1.1; P = .25). Median OS was 8.2 months (95% CI, 7.5 to 8.8) for both treatment arms (HR, 0.9; 95% CI, 0.6 to 1.2; P = .32). COX-2 expression did not independently predict survival. Benefit from celecoxib, restricted to patients with low COX-2 expression, was not significant when adjusted for prognostic factors. CONCLUSION In advanced NSCLC, celecoxib does not improve survival. In this study, COX-2 expression was not a prognostic biomarker and had no predictive value when celecoxib was added to chemotherapy.

MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells

Previously we have described a novel multidrug‐resistant cell line, HT29col, which displayed over expression of the multidrug‐resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172–8). In our study, long‐term screening revealed that, during colchicine‐induced acquisition of multidrug resistance in a new HT29col cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol‐insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1) Resistance was fully inhibited by MK571. 2) GCS expression and activity were not upregulated in HT29col cells. 3) Inhibition of GCS did not affect MRP1‐mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug‐resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival.

Current smoking-specific gene expression signature in normal bronchial epithelium is enhanced in squamous cell lung cancer†

Cigarette smoking is the main risk factor for the development of squamous cell lung carcinoma (SCC). However, the smoking‐related molecular changes in SCC have not been studied. Gene expression studies in both histologically normal bronchial epithelium and SCC epithelial samples identified genes differentially expressed between current and ex‐smokers. Subsequently, expression levels of the smoking‐related genes in normal bronchial epithelium were compared with those in SCC cells, since we hypothesized that the smoking‐induced changes would be also deregulated in SCC. Gene expression profiles were generated using Agilent whole human genome microarrays on laser‐microdissected normal bronchial epithelium and SCC samples. Expression levels of 246 genes, mainly related to oxidative stress response, were significantly different between normal bronchial epithelium of current and ex‐smokers. Such a differential gene expression profile did not exist in SCC cells of smokers and ex‐smokers. Interestingly, when comparing SCC and normal bronchial epithelium from ex‐smokers, the vast majority of these 246 genes were also deregulated in SCC. When comparing SCC with normal epithelium from smokers, 22% of the up‐regulated genes showed a similar high expression in SCC whereas 79% of the down‐regulated genes were even further reduced in SCC as compared to current smokers. The down‐regulated genes included several tumour suppressor genes, such as C9orf9, INHBB, LRIG1, SCGB3A1, SERPINI2, STEAP3 and ZMYND10. Thus, our study shows that the majority of genes up‐regulated in normal bronchial epithelium of current smokers show similar high expression levels in SCC, while down‐regulated genes are even further repressed in SCC. Our data indicate that smoking‐related changes in normal bronchial epithelial cells persist in malignant transformed squamous cells. Copyright

Genomic aberrations in squamous cell lung carcinoma related to lymph node or distant metastasis

About 50% of patients presenting with resectable lung cancer develop distant metastases within 5 years. Genomic markers predicting metastatic behaviour of squamous cell lung carcinoma (SCC) are currently underexposed. We analyzed a cohort of patients with primary SCC using array-based comparative genomic hybridization (aCGH) to identify which genomic aberrations are related to metastatic behaviour. The cohort consisted of 34 patients with a follow-up of at least 5 years, 8 with metastases in regional lymph nodes only and 26 patients without any metastases at the time of surgery. Eleven of the latter 26 developed metastases in distant organs within 3 years after surgery. Copy number changes observed in at least 40% of all SCC included gains at chromosomal arms 3q, 5p, 8q, 19q, 20p, 22q and losses at 3p, 4p, 4q, 5q, 8p and 9p. High copy number amplifications were observed at 2p15-p16, 3q24-q29, 8p11-p12, 8q23-q24, and 12p12, containing candidate oncogenes such as BCL11A, REL, ECT2, PIK3CA, ADAM9, MYC and KRAS. Amplification of 2p15-p16 is a novel finding in SCC. Another novel finding is the homozygous deletion observed at 4q33-34.1 in 15% of the SCC cases. Gains at 7q36, 8p12, 10q22, 12p12, loss at 4p14 and the homozygous deletions at 4q occurred significantly more frequent in SCC from patients with lymph node metastases only. SCC from patients with distant metastases showed a significantly higher gain frequency at 8q22-q24 and loss at 8p23 and 13q21, and a significantly lower gain frequency at 2p12 and 2p16 and loss at 11q25 compared with SCC from patients without metastases. Of these, gains at 7q, 8p and 10q were restricted to SCC with lymph node metastasis and gain at 8q was restricted to patients with distant metastasis. Two genomic aberrations, i.e. loss of 4p and gain of 19q12 were observed more frequently in SCC with only lymph node metastases as compared to SCC with distant metastases. In conclusion, we identified genomic aberrations in primary SCC that were related to lymph node or distant metastases.

Sphingolipid metabolism enzymes as targets for anticancer therapy

Treatment with anti-cancer agents in most cases ultimately results in apoptotic cell death of the target tumor cells. Unfortunately, tumor cells can develop multidrug resistance, e.g., by a reduced propensity to engage in apoptosis by which they become insensitive to multiple chemotherapeutics. Ceramide. the central molecule in cellular sphingolipid metabolism, has been recognized as an important mediator of apoptosis. Moreover, an increased cellular capacity for ceramide glycosylation has been identified as a novel multidrug resistance mechanism. Indeed, virtually all multidrug resistant cell types exhibit a deviating sphingolipid composition, most typically an increased level of glucosylceramide. Thus, the enzyme glucosylceramide synthase, which converts ceramide into glucosylceramide, has emerged as a potential target to increase apoptosis and decrease drug resistance of tumor cells. In addition, several other steps in the pathways of sphingolipid metabolism arc altered in multidrug resistant cells, opening a perspective on additional sphingolipid metabolism enzymes as targets for anti-cancer therapy. In this article, we present an overview of the current understanding concerning drug resistance-related changes in sphingolipid metabolism and how interference with this metabolism can be exploited to over come multidrug resistance.

PDMP sensitizes neuroblastoma to paclitaxel by inducing aberrant cell cycle progression leading to hyperploidy

The sphingolipid ceramide has been recognized as an important mediator in the apoptotic machinery, and its efficient conversion to glucosylceramide has been associated with multidrug resistance. Therefore, inhibitors of glucosylceramide synthase are explored as tools for treatment of cancer. In this study, we used d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol to sensitize Neuro-2a murine neuroblastoma cells to the microtubule-stabilizing agent paclitaxel. This treatment resulted in a synergistic inhibition of viable cell number increase, which was based on a novel mechanism: (a) After a transient mitotic arrest, cells proceeded through an aberrant cell cycle resulting in hyperploidy. Apoptosis also occurred but to a very limited extent. (b) Hyperploidy was not abrogated by blocking de novo sphingolipid biosynthesis using ISP-1, ruling out involvement of ceramide as a mediator. (c) Cyclin-dependent kinase 1 and 2 activities were synergistically decreased on treatment. In conclusion, instead of inducing apoptosis through ceramide accumulation, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol by itself affects cell cycle–related proteins in paclitaxel-arrested Neuro-2a cells resulting in aberrant cell cycle progression leading to hyperploidy. [Mol Cancer Ther 2006;5(3):593–601]