Christine Heinzle

Fibroblast Growth Factor Receptors as Therapeutic Targets in Human Melanoma: Synergism with BRAF Inhibition

Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

Fibroblast growth factor receptor 3-IIIc mediates colorectal cancer growth and migration

Background:Deregulation of fibroblast growth factor receptor 3 (FGFR3) is involved in several malignancies. Its role in colorectal cancer has not been assessed before.Methods:Expression of FGFR3 in human colorectal tumour specimens was analysed using splice variant-specific real-time reverse transcriptase PCR assays. To analyse the impact of FGFR3-IIIc expression on tumour cell biology, colon cancer cell models overexpressing wild-type (WT-3b and WT3c) or dominant-negative FGFR3 variants (KD3c and KD3b) were generated by either plasmid transfection or adenoviral transduction.Results:Although FGFR3 mRNA expression is downregulated in colorectal cancer, alterations mainly affected the FGFR3-IIIb splice variant, resulting in an increased IIIc/IIIb ratio predominantly in a subgroup of advanced tumours. Overexpression of WT3c increased proliferation, survival and colony formation in all colon cancer cell models tested, whereas WT3b had little activity. In addition, it conferred sensitivity to autocrine FGF18-mediated growth and migration signals in SW480 cells with low endogenous FGFR3-IIIc expression. Disruption of FGFR3-IIIc-dependent signalling by dominant-negative FGFR3-IIIc or small interfering RNA-mediated FGFR3-IIIc knockdown resulted in inhibition of cell growth and induction of apoptosis, which could not be observed when FGFR3-IIIb was blocked. In addition, KD3c expression blocked colony formation and migration and distinctly attenuated tumour growth in SCID mouse xenograft models.Conclusion:Our data show that FGFR3-IIIc exerts oncogenic functions by mediating FGF18 effects in colorectal cancer and may constitute a promising new target for therapeutic interventions.

Alternative Splicing of Fibroblast Growth Factor Receptor IgIII Loops in Cancer

Alternative splicing of the IgIII loop of fibroblast growth factor receptors (FGFRs) 1–3 produces b- and c-variants of the receptors with distinctly different biological impact based on their distinct ligand-binding spectrum. Tissue-specific expression of these splice variants regulates interactions in embryonic development, tissue maintenance and repair, and cancer. Alterations in FGFR2 splicing are involved in epithelial mesenchymal transition that produces invasive, metastatic features during tumor progression. Recent research has elucidated regulatory factors that determine the splice choice both on the level of exogenous signaling events and on the RNA-protein interaction level. Moreover, methodology has been developed that will enable the in depth analysis of splicing events during tumorigenesis and provide further insight on the role of FGFR 1–3 IIIb and IIIc in the pathophysiology of various malignancies. This paper aims to summarize expression patterns in various tumor types and outlines possibilities for further analysis and application.

Differential effects of polymorphic alleles of FGF receptor 4 on colon cancer growth and metastasis.

A gly(388)arg polymorphism (rs351855) in the transmembrane domain of the fibroblast growth factor receptor (FGFR4) is associated with increased risk, staging, and metastasis in several different types of cancer. To specifically assess the impact of the polymorphic FGFR4 in colorectal cancer (CRC), we engineered CRC cell lines with distinct endogenous expression patterns to overexpress either the FGFR4(gly) or FGFR4(arg) alleles. The biologic analyses revealed an oncogenic importance for both polymorphic alleles, but FGFR4(gly) was the stronger inducer of tumor growth, whereas FGFR4(arg) was the stronger inducer of migration. An evaluation of clinical specimens revealed that FGFR4 was upregulated in 20/71 patients independent of gly(388)arg status. There was no correlation between the presence of an FGFR4(arg) allele and CRC or polyp risk in 3,471 participants of the CORSA study. However, among 182 patients with CRC, FGFR4(arg)-carriers had a fivefold higher risk of tumors that were stage II or greater. Together, our results established that both allelic forms of FGFR4 exert an oncogenic impact and may serve equally well as therapeutic targets in CRC. One important implication of our findings is that FGFR4(arg)-carriers are at a higher risk for more aggressive tumors and therefore may profit from early detection measures.

Fibroblast growth factor receptor 4: a putative key driver for the aggressive phenotype of hepatocellular carcinoma

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.

Is Fibroblast Growth Factor Receptor 4 a Suitable Target of Cancer Therapy

Fibroblast growth factors (FGF) and their tyrosine kinase receptors (FGFR) support cell proliferation, survival and migration during embryonic development, organogenesis and tissue maintenance and their deregulation is frequently observed in cancer development and progression. Consequently, increasing efforts are focusing on the development of strategies to target FGF/FGFR signaling for cancer therapy. Among the FGFRs the family member FGFR4 is least well understood and differs from FGFRs1-3 in several aspects. Importantly, FGFR4 deletion does not lead to an embryonic lethal phenotype suggesting the possibility that its inhibition in cancer therapy might not cause grave adverse effects. In addition, the FGFR4 kinase domain differs sufficiently from those of FGFRs1-3 to permit development of highly specific inhibitors. The oncogenic impact of FGFR4, however, is not undisputed, as the FGFR4-mediated hormonal effects of several FGF ligands may also constitute a tissue-protective tumor suppressor activity especially in the liver. Therefore it is the purpose of this review to summarize all relevant aspects of FGFR4 physiology and pathophysiology and discuss the options of targeting this receptor for cancer therapy.

Up-regulation of 12(S)-lipoxygenase induces a migratory phenotype in colorectal cancer cells

12(S)-Lipoxygenase (LOX) and its product 12(S)-hydroxyeicosatetraenic (HETE) acid have been implicated in angiogenesis and tumour invasion in several tumour types while their role in colorectal cancer progression has not yet been studied. We have analysed 12(S)-LOX expression in colorectal tumours and found gene expression up-regulated in colorectal cancer specimens for which the pathology report described involvement of inflammation. Using cell line models exposed to 12(S)-HETE or over-expressing 12(S)-LOX malignant cell growth as well as tumour cell migration was found to be stimulated. Specifically, Caco2 and SW480 cells over-expressing 12(S)-LOX formed fewer colonies from sparse cultures, but migrated better in filter-migration assays. SW480 LOX cells also had higher anchorage-independent growth capacity and a higher tendency to metastasise in vivo. Knock-down or inhibition of 12(S)-LOX inhibited cell migration and anchorage-independent growth in both 12(S)-LOX transfectants and SW620 cells that express high endogenous levels of 12(S)-LOX. On the cell surface E-cadherin and integrin-β1 expression were down-regulated in a 12(S)-LOX-dependent manner disturbing cell–cell interactions. The results demonstrate that 12(S)-LOX expression in inflammatory areas of colorectal tumours has the capacity to induce an invasive phenotype in colorectal cancer cells and could be targeted for therapy.

Irinotecan Upregulates Fibroblast Growth Factor Receptor 3 Expression in Colorectal Cancer Cells, Which Mitigates Irinotecan-Induced Apoptosis

BACKGROUND: Irinotecan (IRI) is an integral part of colorectal cancer (CRC) therapy, but response rates are unsatisfactory and resistance mechanisms are still insufficiently understood. As fibroblast growth factor receptor 3 (FGFR3) mediates essential survival signals in CRC, it is a candidate gene for causing intrinsic resistance to IRI. METHODS: We have used cell line models overexpressing FGFR3 to study the receptors impact on IRI response. For pathway blockade, a dominant-negative receptor mutant and a small molecule kinase inhibitor were employed. RESULTS: IRI exposure induced expression of FGFR3 as well as its ligands FGF8 and FGF18 both in cell cultures and in xenograft tumors. As overexpression of FGFR3 mitigated IRI-induced apoptosis in CRC cell models, this suggests that the drug itself activated a survival response. On the cellular level, the antiapoptotic protein bcl-xl was upregulated and caspase 3 activation was inhibited. Targeting FGFR3 signaling using a dominant-negative receptor mutant sensitized cells for IRI. In addition, the FGFR inhibitor PD173074 acted synergistically with the chemotherapeutic drug and significantly enhanced IRI-induced caspase 3 activity in vitro. In vivo, PD173074 strongly inhibited growth of IRI-treated tumors. CONCLUSION: Together, our results indicate that targeting FGFR3 can be a promising strategy to enhance IRI response in CRC patients.

Abstract 1203: Targeting FGFR4 in colorectal cancer cells

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in the regulation of cell proliferation, differentiation and survival as well as tumor development and therefore constitute prominent targets for cancer therapy. Over-expression of FGFR4 has been described in various cancer types and was also related to tumor aggressiveness. Reports concerning the role of FGFR4 in colorectal cancer (CRC) are still under dispute. In our study we found FGFR4 expression to be significantly up-regulated in human CRC tissue compared to normal mucosa. CRC cell line models over-expressing FGFR4 were created by transfecting SW480, HCT116 and HT29 colon carcinoma cells with a FGFR4 expression vector. This resulted in an increase of cell proliferation and colony formation under standard tissue culture conditions and colony outgrowth in soft agar was stimulated. In xenografted SCID mice tumor growth was significantly increased by FGFR4 over-expression. siRNA mediated knock down of FGFR4 in the high FGFR4 expressing cell lines HCT116 and HT29 resulted in decreased cell proliferation, viability and colony outgrowth in 2-dimensional growth assays. Additionally a dominant negative FGFR4 adenoviral construct inhibited 3-dimensional anchorage independent growth and suppressed tumor growth in vivo almost completely. Ongoing experiments analyze the effects of a soluble FGFR4 variant on 2-dimensional and 3-dimensional growth. FGFR4 up-regulation resulted in activation of survival pathways via FRS2 and PI3K leading to phosphorylation of GSK3β and S6. Signaling effects by blockage of FGFR4 dependent signaling via siRNA and the dominant negative adenoviral construct are currently being investigated. Based on our results we identified the FGFR4 as an important oncogene in the carcinogenesis which is involved in the tumor growth regulation of CRC in vitro and in vivo. Consequently FGFR4 should be regarded as a new therapeutic target in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1203. doi:1538-7445.AM2012-1203